oxidative stress and risk of cardiovascular disease ......soy protein, which have also demonstrated...

Oxidative Stress and Risk of Cardiovascular Disease Associated with Low- and High-Monounsaturated Fat

Portfolio Diets

By

Laura Chiavaroli

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Nutritional Sciences University of Toronto

© Copyright by Laura Chiavaroli (2010)

ii

Oxidative Stress and Risk of Cardiovascular Disease Associated with Low- and High-Monounsaturated Fat

Portfolio Diets

Laura Chiavaroli Master of Science

Department of Nutritional Sciences University of Toronto

2010

Abstract

The objective was to assess the effect of a high-monounsaturated fat (MUFA) dietary

portfolio of cholesterol-lowering foods on oxidative stress and cardiovascular risk. Twenty-

four hyperlipidemic subjects followed a very low-saturated-fat therapeutic control diet for 4

weeks after which they were randomized to receive the dietary portfolio, consisting of soy

protein (20g/1000kcal), viscous fibre (10.3g/1000kcal), plant sterols (2-3g) and almonds

(21.5g/1000kcal), in combination with high- or low-MUFA (25.9% and 12.9% MUFA,

respectively) for the next 4 weeks, where MUFA replaced 13.0% of dietary carbohydrate.

On high-MUFA, there were significantly greater increases in HDL-C and apoA1 and

significantly greater reductions in total:high-density lipoprotein cholesterol (total:HDL-C)

ratio and high-sensitivity C-reactive protein (hs-CRP) compared to the low-MUFA dietary

portfolio. In all diets there were significant increases in protein thiols and reductions in

conjugated dienes and thiobarbituric acid reactive substances (TBARS) measured in the

LDL-fraction, however no difference between the high- and low-MUFA diets.

Abstract Word Count: 149

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Table of Contents

Chapter Title Page Abstract.............…………………....……………………………...…………….ii

Table of Contents……………………………………………..….…..………iii-v

List of Abbreviations………………………………….…….…….…..…..…vi-vii

List of Figures…………………………....……………….….….…..………...viii

List of Tables………………………………………………..….……………….ix

1 Introduction…………....…………………………………….……………….1-3 2 Literature Review………………………………….………...….………..……4

2.1 Cardiovascular Disease Risk Factors..….……….………………5

2.1.1 Hyperlipidemia……………..……….…….……….……5-6

2.1.2 Oxidative Stress………...…..………….………………6-7

2.1.2.1 Measures of Oxidative Stress….……….…..7-9

2.1.2.2 Supplemental Use of Antioxidants.…..…..9-10

2.1.3 HDL-C…………………………………......…………10-12

2.2 Foods with cholesterol-lowering and anti-oxidative potential...12

2.2.1 Nuts……………………………………………...……12-13

2.2.2 Soy Protein…………………………………………..13-14

2.2.3. Viscous Fibre………………………………………..14-15

2.2.4 Plant sterols and stanols…………………………….…16

2.2.5 The Dietary Portfolio..............................................17-18

2.2.6 Monounsaturated Fatty Acids…………………...…18-20

3 Hypothesis, Objectives & Rationale……………………………..………..21

3.1 Hypothesis………………………………………………………..………..22

3.2 Objectives………………………………………………………..…...……22

3.3 Rationale……………………………………………………………..…22-23

4 The Effect of Adding Monounsaturated Fat to a Dietary Portfolio of Cholesterol-Lowering Foods in Hypercholesterolemia…………...…..24 4.1 Abstract……………………………………………………………………..25

4.2 Introduction…………………………………………………………………26

iv

4.3 Methods…………………………………………………………………….26

4.3.1 Participants………………………………….……………….26-27

4.3.2 Study Protocol……………………………….………………27-28

4.3.3 Diets…………………………………………..………………28-29

4.3.4 Analyses………………………………………...……………29-30

4.3.5 Statistical Analysis…………………………………...………....30

4.4 Results………………………………………………………............……..30

4.4.1 Participants……………………………………………….….30-31

4.4.2 Blood Lipids and C-reactive Protein………………………31-32

4.4.3 Blood Pressure…………………………………………...……..32

4.4.4 Drop Outs and Adverse Events……………………............…32

4.5 Discussion……………………………………………………………...32-36

4.5.1 Figures …………………………………………………………..…..37-39

4.5.2 Tables……………………………………………....……………..…40-45

5 The Effect on Oxidative Stress of Adding Monounsaturated Fat to a Dietary Portfolio of Cholesterol-Lowering Foods.................................46

5.1 Abstract…………………………………………………………………..…47

5.2 Introduction………………………………………………………………...48

5.3 Methods………………………………………………………….….……..48

5.3.1 Participants……………………………………………....…..48-49

5.3.2 Study Protocol………………………………………….……49-50

5.3.3 Diets…………………………………………………...……..50-51

5.3.4 Analyses………………………………………………..….…51-52

5.3.5 Statistical Analysis…………………………………..….……...52

5.4 Results………………………………………………………………….….52

5.4.1 Participants……………………………………...………………52

5.4.2 Markers of Oxidation…..........…………………….………..52-53

5.4.3 Drop Outs and Adverse Events……………………….…........53

5.5 Discussion………………………………………………..……....…….53-55

5.5.1 Figures……………………………………………………………56

5.5.2 Tables…………………………………………………………….57

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6 Overall Discussion, Limitations and Future Research…..….……….58 6.1 Overall Discussion………………………………………….…………59-62

6.2 Limitations………………………………………………………..…….62-63

6.3 Future Research………………………………………………….……63-64

7 Summary……………………………………………………………………….65

7. Summary………………………………………………………………….…66 8 References……………………………………………..…...........……….67-85

vi

List of Abbreviations

8-OHdG – 8-Hydroxyeoxyguanosine

ABC transporter – ATP-binding Cassette Transporter

AHA – American Heart Association

apoA1 – apolipoprotein A1

apoB – apolipoprotein B

BMI – Body Mass Index

CD – Conjugated Dienes

CE – Cholesterol Ester

CETP - Cholesterol-Ester Transfer Protein

CHD – Coronary Heart Disease

CV – Coefficient of Variance

CVD – Cardiovascular Disease

DASH – Dietary Approaches to Stop Hypertension

DNA – Deoxyribonucleic Acid

DTNB – 5,5’-Dithio-bis 2-Nitrobenzoic Acid

FDA – Food and Drug Administration

HDL – High-Density Lipoprotein

HDL-C – High-Density Lipoprotein Cholesterol

HL – Hepatic Lipase

hs-CRP – high-sensitivity-C-Reactive Protein

IVUS – Intravascular Ultrasound

IL-6 – Interleukin-6

LCAT – Lecithin Cholesterol Acyltransferase

LDL – Low- Density Lipoprotein

LDL-C – Low- Density Lipoprotein Cholesterol

LPL – Lipoprotein Lipase

LRC-CPP – Lipid Research Clinic Coronary Primary Prevention

MDA – Malondialdehyde

MET – Metabolic Equivalent of Tasks

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MUFA – Monounsaturated Fatty Acid

NCEP – National Cholesterol Education Program

NCEP ATP III – National Cholesterol Education Program Adult Treatment Panel III

Ox-LDL – Oxidized Low-Density Lipoprotein

PAF-AH – Platelet-Activating Factor Acetylhydrolase

PON1 – Paraoxonase 1

PUFA – Polyunsaturated Fatty Acid

RCT – Reverse Cholesterol Transport

ROS – Reactive Oxygen Species

S-S – Disulfide Bond

SAS – Statistical Analysis System

SEM – Standard Error Mean

SFA – Saturated Fatty Acid

-SH – Thiol Group

TBARS – Thiobarbituric Acid Reactive Substances

Total-C – Total Cholesterol

TG – Triglycerides

TNF-α – Tumor Necrosis Factor - Alpha

USDA – United States Department of Agriculture

VLDL-C – Very Low-Density Lipoprotein Cholesterol

viii

List of Figures

Chapter 2 Figure 1. The development of atherosclerosis.

Figure 2. Oxidative modification of cellular macromolecules.

Figure 3: Reaction of a thiol group from a sulphur-containing amino acid with DTNB as a

measurement of the amount of unoxidized proteins.

Figure 4: Design of the proposed mechanism of HDL-C lowering.

Chapter 4 Figure 1: Patient flow diagram.

Figure 2: Mean (SE) percentage change from baseline for both treatments for HDL-C,

apoA1, total:HDL-C ratio and apoB:apoA1 ratio.

Figure 3. Mean (SE) percentage change from baseline for both treatments for total-C,

LDL-C, apoB and TG.

Chapter 5 Figure 1. Means of oxidative stress markers on high- and low-MUFA diets.

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List of Tables

Chapter 4 Table 1. Baseline characteristics of randomized study participants.

Table 2: Macronutrient Composition of the Participants' Habitual Diet

Table 3: Macronutrient Composition of the Study Diets

Table 4: Example Menuplan of Diets for 2000kcal

Table 5. Effect of Control, Very Low-Saturated-Fat, Diet on Blood Lipids, C-Reactive

Protein, and Blood Pressure in High- vs. Low-MUFA Groups

Table 6. Effect of High-MUFA and Low-MUFA Dietary Portfolio Treatments on Blood

Lipids, C-Reactive Protein, and Blood Pressure

Chapter 5 Table 1. Effect of Control, Very Low-Saturated-Fat, Diet on Oxidative Markers in High-

vs. Low-MUFA Groups

Table 2. Effect of Low-MUFA and High-MUFA Dietary Portfolio Treatments on Oxidative

Markers

1

1. Introduction

2

1. Introduction Cardiovascular disease (CVD) is the leading cause of death in Canada,

accounting for approximately one-third of all deaths, and is the largest economic

burden of disease (1). For prevention and treatment of CVD, attention to diet

and lifestyle has been continuously growing as Canadians are being encouraged

to follow what has been termed a “heart-healthy diet” (2, 3). Essentially this

means limiting intake of saturated fat, choosing leaner meats, low-fat dairy, and

increasing consumption of fruits, vegetables and whole grains as is

recommended by many organizations, including the American Heart Association

(AHA) and the Heart and Stroke Foundation of Canada (4, 5).

Over the past few decades, specific foods have been looked at for their

potential roles in reducing the risk of CVD by targeting one or more of the

associated risk factors. Elevated blood cholesterol is one such risk factor. It is

estimated that about 40% of Canadians have high blood cholesterol levels (6).

The National Institutes of Health in their Third Report of the Expert Panel on

Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult

Treatment Panel, ATP, III Final Report) encourages the use of therapeutic

dietary options for enhanced reduction of low-density lipoprotein cholesterol

(LDL-C), such as plant sterols and viscous (soluble) fibres (7). These dietary

options have been previously combined with other foods, including almonds and

soy protein, which have also demonstrated cholesterol reduction, into a dietary

intervention termed the dietary portfolio, which has been shown to effectively

reduce cholesterol levels to a similar extent as the therapeutic dose of first-

generation statins (28.6% vs. 30.9%, respectively) compared to a low-fat control

diet (8.0%) (8).

Another risk factor for CVD which has been given much attention is

oxidative stress. Many studies have been conducted on the effects of varying

dietary antioxidants on oxidative stress levels and risk of CVD with conflicting

results, indicating the need for further investigation (9). However, it is possible

that through the consumption of whole foods and the interactions between

vitamins and nutrients that a greater benefit may be observed. Dietary portfolio

3

studies have also shown significant reductions in high-sensitivity C-reactive

protein (hs-CRP) and increases in protein thiols, demonstrating the potential to

improve oxidative and inflammatory status (10-12). In addition to antioxidant-rich

foods, the lipid factor high-density lipoprotein cholesterol (HDL-C) has been

associated with improvements in lipid oxidation, as well as anti-inflammatory and

antithrombotic activity. High HDL-C levels have been associated with reduced

oxidative stress and conversely, low HDL-C has been associated with increases

in oxidation (13-15). Methods known to increase HDL-C levels include increased

dietary fat intake, as well as exercise, smoking cessation and moderate alcohol

intake (16, 17). As a result of recommended dietary restrictions on saturated fatty

acid (SFA) and polyunsaturated fatty acid (PUFA) intake, monounsaturated fatty

acid (MUFA) intake is recommended as the dietary method of increasing HDL-C,

which can be achieved through increased consumption of foods rich in MUFA

such as avocados, high-MUFA oils including olive and canola oil, as well as nuts

and seeds.

In the present study, MUFA was added to an effective dietary portfolio of

cholesterol-lowering foods to determine if further reductions could be made in

CVD risk, particularly by targeting the oxidative and lipid risk factors.

4

2. Literature Review

5

2.1 CVD Risk Factors 2.1.1 Hyperlipidemia Cardiovascular disease (CVD) is a leading cause of death in North America.

Although rates of death due to heart disease have declined by 25% in the past decade,

it still accounts for approximately one third of all deaths in both Canada and America

and is expected to rise due to increasing rates of obesity and diabetes, which are known

risk factors for CVD (6, 18). Other risk factors include hypertension, smoking, oxidative

stress and inflammation, and high blood cholesterol, especially elevated LDL-C. Due to

the prevalence of CVD, organizations exist to continuously evaluate scientific evidence

in order to present clinical guidelines on how best to prevent and treat risk factors. The

National Cholesterol Education Program Expert Panel and the Canadian Cardiovascular

Society have developed treatment recommendations, which are updated regularly with

evidence from emerging trials, in order to control hyperlipidemia and reduce the risk of

heart disease. Their recommendations are based on risk of developing coronary heart

disease (CHD), which is calculated, for example, from an equation developed as a

result of a large epidemiological study, the Framingham Heart Study. The Framingham

predictive risk equation is used to predict 10-year risk of CHD and includes the risk

factors age, total cholesterol (total-C), HDL-C , systolic blood pressure, and cigarette

smoking (19). LDL-C remains the primary lipid target because many studies over the

past 3 decades have demonstrated that through the use of statins, LDL-C levels could

be reduced with a corresponding reduction in risk of CVD (20, 21). One meta-analysis

found that for every 1.0mmol/L decrease in LDL-C, there is a corresponding reduction of

21% and 12% in major cardiovascular disease events and all-cause mortality,

respectively (20). However, not only the quantity, but the quality of the LDL-C plays a

very important role in the risk of CVD since when coupled with an increased oxidative

and inflammatory environment, can result in the development of atherosclerosis, which

is one of the most prevalent forms of CVD (22). Research focused on reducing oxidative

stress has been inconsistent, unlike the majority of research on the other risk factors.

For example, blood pressure reduction has been demonstrated in the successful DASH

(Dietary Approaches to Stop Hypertension) trial and can also be controlled through

6

pharmaceuticals (23). Also, there are many methods available for smoking cessation

and exercise can reduce obesity, which coupled with avoiding excess carbohydrate,

especially simple sugars or choosing foods with low glycemic index (GI), can help in the

management or prevention of diabetes (24, 25). Research, however, on reducing

oxidative stress, including the trials on antioxidant supplementation, is still very

controversial, therefore requires further investigation.

2.1.2 Oxidative Stress Oxidative stress occurs when there is an imbalance between the production of reactive

oxygen species (ROS), which occurs naturally from oxidative metabolism, and

antioxidant defences (26). ROS are unstable molecules, such as superoxide, hydroxyl

and hydroperoxyl radicals, with an unpaired electron which can readily extract electrons

from other molecules, thus behaving as oxidants (27). They can act beneficially by

killing pathogens through phagocytes in the immune system, but can also elicit cytotoxic

effects (28). When the amount of ROS rises above normal levels, which has been

associated with many chronic disease states including hypercholesterolemia and

atherosclerosis, the antioxidant system of the body is overwhelmed, resulting in the

oxidation of particles such as proteins and lipids (29). The oxidation of the lipid particle

LDL-C is known to increase its atherogenicity since oxidized LDL (ox-LDL) is

recognized by macrophages and taken up via scavenger receptors. The macrophages

are then thought to become foam cells which form plaque in the arteries and thus, the

development of atherosclerosis (22, 30, 31) (Figure 1). Therefore, the oxidative

modification of LDL-associated lipids is directly involved in the initiation process of

atherosclerosis (32).

7

Figure 1. The development of atherosclerosis (33). Abbreviations: LDL, Low-density

lipoprotein; MM-LDL, minimally modified low-density lipoprotein; ox-LDL, oxidized-low-

density lipoprotein; ROS, reactive oxygen species; SR-A, scavenger receptor A.

2.1.2.1 Measures of Oxidative Stress Due to the very short half-lives of ROS, damage is measured via causally

associated molecules such as those resulting from protein and lipid oxidation (34-37)

(Figure 2).

8

Figure 2. Oxidative modification of cellular macromolecules (36).

Protein oxidation is measured by the oxidation of side chains. The DTNB [5’, 5’-

dithio-bis(2-nitrobenzoic acid)] assay is a common method used to measure the

9

oxidation of sulphur containing amino acid side chains (cysteine and methionine)

because the DTNB molecule reacts with the thiol groups (-SH) (Figure 3). When there is

increased protein oxidation, less thiol groups will be detected since they will be already

oxidized into disulfide bonds (S-S).

Figure 3: Reaction of a thiol group from a sulphur-containing amino acid with

DTNB as a measurement of the amount of unoxidized proteins (38).

For lipids, the oxidation of LDL is of great interest because of its association with

the development of atherosclerosis. There are multiple ways of measuring oxidized

LDL, two of which include via conjugated dienes (CD) and thiobarbituric acid reactive

substances (TBARS) in LDL extracts precipitated from serum. In the former, the amount

of CD is measured directly by spectrophotometric determination of CDs in the extracted

LDL lipids (39). In the latter, the lipid peroxidation product malondialdehyde (MDA) is

measured through its reaction with thiobarbituric acid, which is added to the serum, and

also read spectrophotometrically (40). Considering the association between ox-LDL and

disease states including hypercholesterolemia, diabetes and the development of

cardiovascular disease (41, 42), many studies have investigated the role of antioxidants

in minimizing oxidative damage and reducing the risk of CVD.

2.1.2.2 Supplemental Use of Antioxidants Studies investigating the supplemental use of antioxidants have demonstrated

conflicting results. Some studies have demonstrated reductions in oxidative damage

resulting from antioxidant use, such as a reduction in ox-LDL with high doses of vitamin

E supplementation (43-46). However, most large trials conducted with antioxidant

supplementation have found no effect, such as the MRC/BHF (Medical Research

Council and British Heart Foundation) Heart Protection Study which assessed long term

supplementation of vitamin E, C and beta-carotene daily in subjects at high risk of CHD

(47). Other studies have demonstrated harmful effects, such as the increased risk of

10

lung cancer and cardiovascular disease in smokers supplemented with b-carotene (48),

or the HOPE (Heart Outcomes Prevention Evaluation) Study which found an increase in

heart failure in high risk subjects given 400IU of vitamin E daily for a mean of 7 years

(49). It must be questioned, however, whether these studies were targeting the

oxidative stress risk factor in the appropriate way. It is known that antioxidants may

become pro-oxidants in an oxidative environment, therefore, instead of giving single

vitamin supplements to high risk subjects who are under high oxidative stress, it may be

more effective to target the environment itself as a whole. This may be accomplished by

consumption of foods rich in vitamins in their whole form as it may be through the

interaction of all food components where protection seen in epidemiological studies may

be attained. Some support for this idea includes the results of the DASH trial which

tested a diet rich in fruit, vegetables, whole grains and low-fat dairy and not only

demonstrated significant reductions in blood pressure, but in further analyses, found

significant reductions in markers of oxidative damage (23, 50, 51). Additionally, the main

sources of many vitamins are plant foods, as well as nuts, seeds, and vegetable oils for

vitamin E, therefore, the benefits noted in epidemiological studies may be the result of

multiple vitamins working together or in combination with other nutrients present, such

as phytochemicals, fibre, or polyunsaturated and monounsaturated fats (52). Thus,

further investigation into the consumption of whole foods rich in vitamins and nutrients is

required to allow for possible synergistic activity and the potential promotion of a less

oxidative environment and ultimately, reduction in CVD risk.

2.1.3 HDL-C

Risk factors other than LDL-C are increasingly receiving attention due to the

residual risk for CVD that remains once LDL-C has been effectively lowered. This

includes the growing area of research on low HDL-C and risk of CVD independent of

LDL-C, total-C and TG. Many studies have found that for every 0.026mmol/L increase in

HDL-C, there is a corresponding 1-3% reduction in CVD risk and that even when LDL is

reduced or total-C levels are low, those with lower HDL-C are at a much greater risk of

developing CVD (53-61). As a result of the inverse correlation between HDL-C levels

and risk for CVD, the activities of HDL-C have been investigated. HDL-C is known for its

11

facilitating role in reverse cholesterol transport (RCT) whereby excess cholesterol from

the peripheral tissues is transferred from the plasma to the liver where it is either

recycled or excreted from the body through bile (62). Additionally, HDL-C has many

effects on the endothelium and antithrombotic actions (63, 64). However, its ability to

inhibit the oxidative modification of LDL via its potential antioxidative activity may be of

greater interest since oxidized LDL plays a central role in the initiation and propagation

of atherosclerosis (65-67). Mechanistically, there are many possible pathways by which

HDL-C may inhibit LDL oxidation, such as through the HDL-associated proteins

paraoxonase 1 (PON1) (14, 68) and platelet-activating factor acetylhydrolase (PAF-AH)

(69). The former protects against LDL oxidation, reverses biological effects of oxidized

LDL, inhibits the oxidation of HDL thereby preserving its function, and has been

associated with reduced risk of CVD (13, 70-75). The later attenuates proinflammatory

activity of PAF and functions as an antioxidant by hydrolyzing oxidized phospholipids

which result from the oxidation of LDL (76-78).

Another important component of HDL-C is its associated apolipoprotein, apoA1.

ApoA1 is a useful clinical measure because it can be measured directly with little error

and is a reflection of the anti-atherogenic HDL-C. It plays a central role in RCT, as it is

responsible for its initiation by picking up cholesterol from the periphery and delivering it

to the liver in HDL particles, and it has also been shown to have anti inflammatory and

antioxidant effects (79). Recently, apoA1, as well as the apolipoprotein associated with

LDL-C, apoB, have been called more informative risk indicators than their lipoproteins

or other lipids (79, 80). Two very large epidemiological studies, the AMORIS

(Apolipoprotein Mortality RISk) and INTERHEART studies (A Global Study of Risk

Factors for Acute Myocardial Infarction) found that there was a very strong direct

relation between the apoB:A1 ratio and fatal or acute myocardial infarction and that it

was the strongest lipid ratio predictor of CVD risk (81, 82). Interest in apoA1 began in

the early 1980s when it was discovered that a small group of people in Italy carried a

mutation of the apoA1 gene that resulted in low HDL-C and apoA1 and high TG but low

rates of CVD, which they termed apoA1Milano (83, 84). Studies began in the 1990s with

the recombinant version of apoA1 and those in animals demonstrated regression of

atheroma volume and promotion of more stable plaques (85-87). A few studies have

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been conducted in humans also demonstrating significant regression of atherosclerosis

when given recombinant apoA1Milano complexed with phospholipids to mimic properties

of nascent HDL (85, 88). It appears that the mutation results in a form of apoA1 with

elevated performance of the activities of normal apoA1. Additionally, a number of

prospective cohort studies have concluded that HDL-C and apoA1 are strong predictors

of CVD risk, with some suggesting apoA1 is the stronger predictor (58, 89-95). As a

result, some countries have expanded their clinical guidelines for decreasing CVD risk

to include apoA1, including Sweden and Norway (96). Therefore, apoA1 and HDL-C

remain potential targets for anti-atherosclerotic therapeutic strategies. In order to test whether elevation in HDL-C can improve CVD risk, methods of

increasing HDL-C have been elucidated. Those that have demonstrated success

include exercise, smoking cessation, weight loss, moderate alcohol intake and

increased MUFA consumption (16, 17, 97-99) and they have also demonstrated the

potential to reduce CVD risk (100-104). From a dietary approach, MUFAs could be

easily incorporated into the diet as they are found naturally in oils, such as olive and

canola, as well as in nuts, seeds and avocados.

2.2 Foods with cholesterol-lowering and anti-oxidative potential 2.2.1 Nuts The many health benefits of nuts have fuelled investigations into their effects on

CVD risk with many having an associated decreased risk (105-109). Some studies

indicated that a CVD risk reduction of 35% or more may be obtained with increased nut

consumption (≥ 2-5 servings per week) (110-112). As a result, nuts have been permitted

a health claim for heart disease risk reduction by the FDA stating that as part of a diet

low in saturated fat and cholesterol, consumption of 42g (1½ oz) of almonds per day

may reduce the risk of heart disease (113). In addition to studies looking at the overall

effect of nut consumption on CVD risk, many studies have investigated potential ways

by which nuts target risk factors, including effects on lipids.

LDL-C has been shown to be significantly reduced by the consumption of nuts,

primarily almonds and walnuts (114, 115). Consumption of between 68 to 100g per day

13

of almonds has been shown to result in LDL-C reductions of 7 to 29% (116, 117). Even

consuming a handful (approximately 1 ounce) of almonds a day has demonstrated

reductions in LDL-C around 4-5% (118). Almonds are an excellent source of the anti-

oxidant vitamin E, phytochemicals, flavonoids, fibre, protein, and MUFA, however

exactly which part of the nut results in the lipid benefit is unknown. Some studies

comparing the effects of the whole nut to the oil from the nut have demonstrated similar

reductions in total- and LDL-C, therefore there is the potential for the effect to arise from

the type of fats found within the almond (115), most of which is MUFA. In addition to the

lipid lowering effects, almonds have associated with a reduction in oxidative damage.

The antioxidant activity of almonds has been demonstrated in reductions in LDL

oxidizability, inflammatory molecules and endothelial dysfunction (119). Acute studies

have demonstrated that whole almonds decreased the susceptibility of lipids to

oxidation and increased the total antioxidant capacity, and did so more than compared

to walnuts (120). Additionally, studies investigating the effects of the skins of almonds

have demonstrated that the variety of polyphenols within have anti-oxidant activity

against specific radicals (121, 122). Due to the fact that the different components of

almonds have potential antioxidant activity, greater oxidative reduction may result from

the consumption of whole almonds thereby targeting a larger variety of radical types

and the health conditions that may arise from each.

2.2.2 Soy Protein

The effect of soy protein on blood cholesterol has been of interest since the early

1900s when the idea emerged that animal protein had negative consequences in the

arteries and thus a search for a plant protein replacement began (123). Compared to

lean meat protein, some studies have demonstrated soy protein significantly reduced

LDL-C around 10% versus 5%, suggesting a specific effect of proteins on cholesterol

transport and metabolism (123, 124), whereas other studies have reported significant

reductions of only 5-6% (125-131). Studies comparing the effects of soy protein to

casein protein express similar results where soy protein effective reduced LDL-C 16%

more than did the casein protein treatment (132). A meta-analysis conducted in 1995

indicated a 12.5% reduction in LDL-C as a result of an average 47g per day of soy

14

protein whereas subsequent studies did not support as great a reduction (126, 133-

135). However, in an article by Sirtori in 2007, who conducted many of the early studies

expressing the beneficial effects of soy protein, he re-evaluated the more recent studies

using a ‘nomogram’ prepared on the basis of initial cholesterol concentrations in the

1995 meta-analysis and concluded that the studies done in the past 10 years are in

agreement with the conclusion of that meta-analysis (136). Additionally, a recent meta-

analysis by Harland concluded that modest soy protein intakes of around 25g per day,

almost half as much as the 1995 meta-analysis, resulted in highly significant reductions

in LDL-C of 6%, which supports the previous meta-analysis (131).

There has been much debate over the effectiveness of soy protein versus soy

isoflavones, and over the effectiveness of highly processed soy products. However, soy

isoflavones have not acquired as much support for their cholesterol-lowering effects and

there are many studies indicating the greater the processing of soy products, the less

effective in cholesterol reduction (133-135, 137, 138). Therefore, the most effective

method of cholesterol-reduction with soy foods is by using whole soy foods which are

least processed.

Soy protein also has potential for reducing lipid oxidation. Many studies done in

animals (139-144) and in humans (145, 146) have reported significant antioxidant

activity resulting from soy protein consumption, possibly by up-regulating LDL-receptor

machinery in the liver, resulting in decreased LDL levels and LDL oxidation. Studies

have demonstrated about a 9-16% reduction in conjugated dienes in the LDL fraction

with intakes of soy foods of around 33g/d (147, 148) Again, there is debate whether it is

the soy isoflavones that contribute the antioxidant properties, however in this respect,

there is more evidence supporting its role (149, 150) in addition to those on whole soy

foods (147, 148). Overall, epidemiological studies suggest the soy consumption is

associated with a reduced risk of CVD (151).

2.2.3 Viscous Fibre

Dietary fibres can be classified based on their solubility. The insoluble fibres include

lignins and cellulose, whereas soluble fibres, also known as viscous fibres, include

natural gel-forming fibres such as pectins, gums and mucilages. Overall, dietary fibre is

15

associated with reduced risks for many diseases including many cardiovascular

diseases (152, 153). However, much of the attention on fibres is on the soluble fibres

including oats, pectins, guars and psyllium and their LDL-C lowering abilities. Common

foods high in viscous fibre include whole oats, barley, psyllium powder, oat bran,

eggplant, and okra. Many studies have demonstrated reductions of 7-9% in LDL-C and

that intakes of only 3g of soluble fibre can have significant reductions in LDL-C of

0.13mmol/L (154) and the consumption of fibre from 3 apples or 3 bowls (28g) of

oatmeal can result in total-C reductions of 2% (154, 155). There are a few possible

mechanisms of effect including increased bile acid output resulting in less reabsorption

of cholesterol and greater usage of cholesterol for hepatic bile acid synthesis (156,

157), inhibition of hepatic fatty acid synthesis by products of fermentation (158), and

changes in intestinal motility (159). Studies of soluble fibre have also shown reductions

in small LDL subfractions which are associated with more atherogenic conditions (160).

In addition to the cholesterol-lowering effect of viscous fibres, other

cardiovascular benefits include reductions in the inflammatory marker CRP (161, 162)

and blood pressure (163, 164) and some studies have demonstrated the potential for

antioxidant activity. Animal studies have demonstrated guar gum can increase

antioxidant protein expression and therefore decrease oxidative stress-induced arterial

injury (165). Also, since oxidative stress can result from inflammation, the anti-

inflammatory potential of fibre is also of interest. In the Iowa Women’s Health Study,

whole grain consumption was associated with a reduced risk of death attributed to

inflammatory diseases which were noncardiovascular of >35% for those who reported

the highest intake (≥ 22.5 servings per week) and since whole grains are an excellent

source of fibre, there is potential for fibre to play a role in reducing inflammation (166).

The same group analyzed the total antioxidant capacity in over 1000 food samples

obtained from the US Department of Agricultural National Food and Nutrient Analysis

Program and found that several whole grain products were at the top of the ranked list

(166). This may be the result of increased enterlactone concentrations associated with

whole grain intake, as a product of lignin precursors within whole grains, which have

anti-oxidative activity (166).

16

Overall, the multifactorial effects of viscous fibre and its large range availability

make it an attractive addition to obtain greater cardiovascular health.

2.2.4 Plant Sterols and Stanols Plant sterols and stanols are structurally similar to cholesterol with sterols being

found naturally in plant foods such as vegetable oils, nuts and seeds, and stanols being

commercially available hydrogenated sterols (167). They are believed to exert their

cholesterol-lowering abilities by decreasing the absorption of dietary and biliary

cholesterol, thus increasing cholesterol synthesis and the activity of LDL-receptors and

ultimately reducing serum LDL-C concentrations (167, 168). Studies have

demonstrated these effects over the past 40 years with plant sterols/stanols enriched in

vehicles such as margarines, dairy products and orange juice and have shown that for

consumption of 3.4g of esterified plant sterol/stanol (or 2.1g plant sterol/stanol)

reductions in LDL-C range from 5 to 16% (169-172), with margarines, the more

common vehicle, averaging about 11% reduction (169). As a result, the United States

Food and Drug Administration (FDA) granted plant sterols health claim status for CHD

risk reduction (113) and NCEP ATP III recommends them for cholesterol reduction (19).

Although there have been some studies questioning the safety of plant sterols (173-

177), many studies have alleviated those concerns (178-182) and a recent review

assessing their efficacy and safety concluded that they are important for cholesterol-

lowering and good heart health (183).

There is also some evidence for potential anti inflammatory and antioxidative

effects of plant sterols. Some studies have shown they may inhibit the secretion of

inflammatory mediators, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-

α), although most of these studies have been done in animals (184). The studies on

their antioxidative effects have been done in humans and demonstrate a reduction in

ox-LDL with 2-3g plant sterol consumption, thus suggesting the potential to protect LDL

from oxidation (185, 186). However, some studies have shown no effect (187).

As a result of their cholesterol-lowering ability and the potential for anti

inflammatory and antioxidative activity, plant sterols may contribute to reducing CVD

risk.

17

2.2.5 The Dietary Portfolio

In the late 1990s with the growing interest in diet in the management of elevated

blood lipids and the many studies investigating the cholesterol-lowering effects of

particular foods, many organizations began recognizing key foods which had

demonstrated effectiveness in cholesterol reduction. The NCEP ATP was

recommending 2g/d of plant sterols and 10-25g/d of viscous fibre (19) and the AHA was

encouraging the consumption of soy proteins (188) for the management of elevated

cholesterol. The FDA had also approved health claims for plant sterols (113), viscous

fibres (189) and soy protein (190). Mechanistically, studies had shown that viscous

fibres increased bile acid losses (191-193), plant sterols increased fecal cholesterol

losses (194, 195) and soy proteins decreased hepatic cholesterol synthesis and

increased LDL-receptor-mediated cholesterol uptake (196, 197). With this information,

Dr. David Jenkins saw the potential for greater cholesterol reduction through dietary

means and therefore combined these foods into what he termed the “dietary portfolio”.

In a preliminary study combining 1.2g/1000kcal plant sterols, 8.3g/1000kcal viscous

fibres, and 16.2g/1000kcal soy protein, as well as 2.9g/1000kcal of almonds as an

added source of vegetable protein and because it also had previously demonstrated the

potential for cholesterol reduction (114), Dr. Jenkins found this dietary portfolio had an

additive effect in lowering LDL-C by 29% (198). With these results, and an FDA health

claim for nuts which encouraged an increase in the contribution of almonds to

16.2g/1000kcal in the dietary portfolio, a series of studies assessing the many benefits

of this dietary portfolio began (8). When tested alongside a first generation statin, the

dietary portfolio demonstrated LDL-C reductions of a similar magnitude (30.9% and

28.6%, respectively), as well as reductions in hs-CRP (33.3% and 28.2%, respectively)

(10). When assessed for long-term effects under real-world conditions, as opposed to

metabolically controlled, 14% and 12% reductions in LDL-C were seen at 3 months and

1 year, respectively, with >30% of subjects maintaining a reduction >20% (199).

Additionally, significant reductions in blood pressure (200), haematological indicies

18

(201) and reductions in the smallest and most atherogenic subclass of LDL-C (202)

have also been demonstrated over the years. Most recently, strawberries were added to

the dietary portfolio and demonstrated significantly improved palatability and reductions

in TBARS as a measure of oxidative stress, while maintaining reductions in blood lipids,

therefore suggesting that added berries may improve the overall utility of the diet (12). It

is thus of interest to continue to explore methods of improving this effective cholesterol-

lowering diet to further reduce the risk of CVD.

2.2.6 Monounsaturated Fatty Acids (MUFAs) With interest in determining the best replacement for SFA, many studies have

looked into the effect of MUFA compared to carbohydrates and PUFA. In terms of

effects on blood lipids, all three of the above food components are effective in

cholesterol reduction when compared to the average American diet (203, 204).

However, when looking into the relative effects on markers of oxidation, many studies

have demonstrated a significant difference with MUFA being the better choice.

MUFA consumption has been found to result in less oxidative modification of

LDL-C compared to when PUFAs are consumed (205), and additionally, when

carbohydrate is replaced by MUFA there are reductions in such measurements of

oxidation as LDL oxidation lag phase and TBARS (203, 206, 207). There are also many

studies which conclude that MUFAs are the preferred substitution due to a greater

potential risk reduction for CVD (203, 208). The AHA also indicates in its statement that

there are prospective observational studies which document that diets rich in MUFAs

are associated with a decreased risk of CHD (100).

MUFAs are most commonly found in oils such as olive and canola, as well as in

nuts, seeds and avocados. Many studies on olive oil on risk of CVD have demonstrated

reductions in markers of oxidative stress. One study which fed participants olive oil at

real-life doses (25mL/day), found a significant increase in the oleic/linoleic acid ratio in

LDL-C and that with every 1 unit increase in the ratio, there was an associated

decrease of 4.2ug/L in plasma isoprostanes, an in vivo measure of ox-LDL (209). Other

studies have also demonstrated how an increase in the amount of oleate in the diet can

reduce the susceptibility of LDL to oxidative modification (210).

19

There is also interest in MUFA because of the potential to raise HDL-C, which is a

risk factor for CVD when levels are low. Although some studies have found no effect of

increased MUFA consumption on HDL-C levels (211-214), there are many studies

which provide support for their potential (203, 215, 216). In the early 1970s when the

interest in HDL-C as a cardioprotective factor began and the search for a suitable

replacement for SFA was of interest, large quantities of PUFA (as much as 20 to 30% of

total calories) were demonstrated to result in great reductions in HDL-C along with LDL-

C, whereas MUFA replacements reduced LDL-C while preserving HDL-C (207, 217).

Studies trying to explain these results compared the different fatty acids to carbohydrate

and found that each had HDL-C raising effects relative to carbohydrate, with PUFAs

having the least and SFA having the greatest with MUFA closely following (218, 219).

Further investigations demonstrated that the decreases in HDL-C and apoA1 were

associated with increased fractional clearance of apoA1, which was later supported by

further evidence showing decreased HDL-C was associated with reduced apoA1

production in addition to increased apoA1 clearance (220, 221). Therefore it is possible

that MUFA may raise HDL-C by increasing secretion of apoA1 or reducing its clearance.

Investigation into the kinetics of HDL-C and apoA1 suggest possible mechanisms

by which these levels are affected. These began as a result of many negative

correlations observed clinically between plasma TG levels, HDL-TG content and fasting

plasma HDL-C and apoA1 concentrations (222-226). Studies have demonstrated that

the metabolism of HDL-C is affected by the interactions between TG-rich lipoproteins

and HDL-C in plasma in at least two ways; firstly, through the lipolytic enzyme

lipoprotein lipase (LPL) which breaks down TG-rich lipoproteins forming surface

materials transferred to HDL, impacting its maturation (227-229), and secondly, through

the cholesterol ester transfer protein (CETP) which transfers TGs from TG-rich

lipoproteins to HDL in exchange for cholesterol esters (CE) producing TG-rich, CE-poor

HDL which is more prone to catabolism (230-232). Kinetic studies in humans with

hypertriglyceridemia and low HDL-C have demonstrated that there is a significant

increase in the fractional catabolic rate of apoA1 but no reduction in apoA1 production

rates, therefore it is the enhanced clearance of HDL-C and apoA1 rather than a reduced

production of HDL-C resulting from LPL which may contribute to the reduced

20

concentration of HDL-C (233-237). However, authors of these studies note that the

CETP-mediated lipid exchange process itself can reduce plasma HDL-C but must be

coupled with additional processes in order to reduce apoA1 concentrations which

include the following: 1. TG-rich, CE-poor HDL are thermodynamically less stable and

have the apoA1 more loosely bound, 2. TG-rich, CE-poor HDL are more readily

lipolyzed by hepatic lipase (HL), which reduces HDL size and releases apoA1, 3.

smaller HDL are more readily cleared from circulation (Figure 4). Since CETP is highly

dependent on the concentration of TG-rich lipoproteins in the plasma, it is possible that

by decreasing the concentration of TG in the circulation through decreasing glucose or

carbohydrate intake by replacement of MUFA, HDL-C and apoA1 concentrations can be

preserved by the converse of this proposed mechanism.

Figure 4: Design of the proposed mechanism of HDL-C lowering (238).

Therefore, increased MUFA consumption by replacement of carbohydrate, may

decrease oxidative stress directly through free radical quenching as supported in

studies where less oxidation is observed when SFA is replaced by MUFA compared to

replacement by PUFA, or indirectly through reductions in glucose fluctuations and thus

TG production, potentially preserving HDL-C along with its antioxidative properties.

21

3. Hypothesis, Objectives, and Rationale

22

3.1 Hypothesis Increasing the dietary intake of MUFA will increase levels of HDL-C compared to

the lower fat, higher carbohydrate diet, and decrease oxidative stress through reduced

ox-LDL and increased protein thiols indicative of a protection of plasma proteins.

3.2 Objectives Overall Objective: To assess the effect on serum lipids and oxidative damage to

proteins and lipids of the replacement of carbohydrate with MUFA in

a cholesterol-lowering diet.

1. To determine the effect of a cholesterol-lowering dietary

portfolio combined with either high- or low-MUFA on HDL-C and

apoA1.

2. To determine the effect of a cholesterol-lowering dietary portfolio

in combination with either a high- or low-MUFA on oxidative

damage to protein and lipids, as assessed by protein thiols and

CD and TBARS in the LDL fraction, respectively.


combined with either high- or low-MUFA on LDL-C and apoB.


combined with either high- or low-MUFA on the total:HDL-C ratio

and the apoB:A1 ratio.

3.3 Rationale The dietary portfolio is a combination of foods which have been individually

researched as effective in blood lipid control and to have some antioxidant activity.

Previous dietary portfolio studies have consistently demonstrated significant reductions

in LDL-C (10, 198, 199, 239). Recently, there is increasing concern in the risk factors for

CVD other than LDL-C due to the residual risk that remains once LDL-C has been

effectively reduced. One such risk factor includes low HDL-C which is present in over a

third of adult men and women. Studies have suggested that for every 0.026mmol/L

increase in HDL-C, there is a corresponding 1-3% decrease in CVD risk (53-55, 97).

23

There is much evidence that both MUFA and HDL-C have antioxidative potential

and that the former may increase the later (13, 66, 71, 203, 218, 219, 240, 241). MUFAs

can easily be incorporated into the diet through the use of high-MUFA oils including

olive and canola oil, as well as avocados, nuts and seeds.

It therefore is appropriate to combine a high-MUFA background diet to an already

effective cholesterol-lowering diet to further reduce the risk of CVD.

24

4. Study: The Effect of Adding Monounsaturated Fat to a Dietary Portfolio of Cholesterol-Lowering

Foods in Hypercholesterolemia

25

The Effect of Adding Monounsaturated Fat to a Dietary Portfolio of Cholesterol-Lowering Foods in Hypercholesterolemia

4.1 Abstract

Background: Higher monounsaturated fat intakes may raise HDL-C without raising

LDL-C. We have therefore tested whether increasing the monounsaturated fat (MUFA)

content of an effective LDL-C lowering diet (dietary portfolio) both increases HDL-C and

further reduces the total:HDL-C ratio, two key risk factors for CVD.

Methods: Twenty-four hyperlipidemic participants took a very low-saturated-fat

therapeutic diet for one month and then were randomized to a low- or high-

monounsaturated-fat dietary portfolio for a further month. Food was provided for the 2

month period with calorie intake based on Harris-Benedict estimates for energy

requirement.

Results: On the high-MUFA dietary portfolio, HDL-C rose 13.1±3.6% (P=.004),

whereas on the low-MUFA dietary portfolio, HDL-C did not change (2.7±3.6%, P=.466)

(treatment difference, P=.004). The respective figures for the total:HDL-C ratio were -

24.5±2.1% (P<.001) on high-MUFA versus -17.6±3.0% (P<.001) on low-MUFA

(treatment difference, P=.006). These treatment differences were associated with

significantly higher apoA1 concentrations on high-MUFA dietary portfolio. CRP was also

significantly reduced on the high-MUFA dietary portfolio. No treatment difference was

seen in body weight although a mean weight loss was seen over both low-MUFA (-0.98

± 0.26kg) and high-MUFA (-0.78 ± 0.21kg) diets.

Conclusions: Monounsaturated fat increased the effectiveness of a cholesterol-

lowering dietary portfolio and may reduce cardiovascular risk through raising HDL-C,

further lowering the ratio of total:HDL-C and also by reducing CRP.

26

4.2 INTRODUCTION

Strategies that combine cholesterol-lowering foods or food components such as

viscous fibres and plant sterols have been recommended to enhance the effectiveness

of therapeutic diets low in saturated-fat and cholesterol (19, 242). Such dietary

combinations (dietary portfolio) have resulted in substantial reductions in LDL-C (10)

and its apolipoprotein, apoB, but the effects on HDL-C and its apolipoprotein, apoA1,

have been less apparent (199). Low plasma HDL-C and apoA1 concentrations and an

elevated ratio of total:HDL-C are recognized risk factors for CVD (243-247). Thus,

dietary strategies that both lower total and LDL-C and raise HDL-C should have broad

application. One method for increasing HDL-C appears to be the use of

monounsaturated fat (MUFA), particularly when MUFA replaces dietary carbohydrates

(248, 249). Furthermore, increased intakes of MUFA through increased nut

consumption and vegetable oil intake has been associated with a reduced incidence of

CVD in cohort studies (250, 251).

We have therefore compared the effect on serum lipids of substituting 13.0% of

total calories as carbohydrate for MUFA in a dietary portfolio that has been shown to be

effective under controlled conditions in lowering LDL-C by 28% and the total:HDL-C

ratio by 24% (10).

4.3 METHODS

4.3.1 Participants

Men and women with mild to moderate hypercholesterolemia were recruited from

the Clinical Nutrition and Risk Factor Modification Centre at St Michael’s Hospital,

Toronto, Ontario, and from newspaper advertisements. Only postmenopausal women

were recruited because of the increase in LDL-C and CHD risk in women of this age

and to avoid possible fluctuations in blood lipids related to the menstrual cycle.

Participants were selected who had previously demonstrated LDL-C levels >4.1 mmol/L

(19, 242). No participants were recruited if they had a history of cardiovascular disease,

untreated hypertension (blood pressure >140/90mmHg), diabetes, or renal or liver

disease, or were taking medications known to influence serum lipids apart from stable

doses of thyroxine. All medications and supplements were expected to be taken at a

27

constant dose prior to and during the study. Iron supplementation, ferrous gluconate

7mg tid (Salus-Haus, Bruckmuhl, Germany), was provided to participants on the basis

of a prestudy ferritin of <50mcg/L.

4.3.2 Study Protocol The study followed a randomized parallel design and was carried out between

August 2007 and April 2009. Participants were counselled to follow their own low-

saturated- fat therapeutic diets for 1 month prior to the start of the study. They were

then provided with a very low-saturated-fat dairy and whole-grain cereal metabolically

controlled diet which acted as a stabilization period prior to the high- and low-MUFA

diets. After one month on this metabolically-controlled diet, they were randomized to

take either a high-MUFA or conventional (low-MUFA) dietary portfolio for a final month.

Throughout the study participants attended weekly clinic visits where body weight

was taken and blood pressure was measured three times in the non-dominant arm

using an automated digital blood pressure monitor (OMRON Healthcare Inc, Vernon

Hills, Illinois) by the same observer. At 2-week intervals blood samples were obtained

after 12-hour overnight fasts. A seven-day diet history was obtained for the week prior

to the 2-month metabolic treatment period. Completed menu checklists were returned at

weekly intervals, as well as 7-day exercise records, during the 4-week diet periods and

checked by the dietitians, who also checked the participants’ exercise and ensured that

it was constant over the course of the study period. Exercise was calculated as

metabolic equivalent of tasks (METs) (252).At weekly intervals, participants recorded

their overall feeling of satiety using a 9-point bipolar semantic scale in which −4 was

extremely hungry, 0 was neutral, and +4 was uncomfortably satiated.

Participants were randomized to the low- and high-MUFA portfolio diets and

stratified on the basis of sex and an LDL-C of <4.2mmol/L or >4.2mmol/L during the first

2 weeks of the 4 weeks of the very low-saturated-fat therapeutic control diet by the

statistician using a random number generator and SAS version 9.1 software (SAS

Institute Inc, Cary, NC) (253) in a separate location from the clinic. The dietitians were

not blinded to the diet because they were responsible for patients’ diets and for

checking diet records. The laboratory staff responsible for analyses were blinded to

28

treatment and received samples labelled with name codes and dates. The study was

approved by the ethics committees of the University of Toronto, St. Michael’s Hospital

and the Natural Health Products Directorate of Health Canada. Written informed

consent was obtained from all participants. Participants were offered no financial

compensation for participation in the study. The clinical trial registration number is

NCT00430430.

4.3.3 Diets

The diets eaten before the 8-week study were the participants’ routine

therapeutic low-fat diets, which were similar to current National Cholesterol Education

Program guidelines (<7% energy from saturated fat and <200 mg/d of dietary

cholesterol) (19) and previously referred to as a Step II diet (248) (Table 2). During the

8-week study period, diets were provided based on estimated caloric requirements

using the Harris-Benedict equation. Foods were sourced from Loblaws supermarket in

Toronto and health food stores. All diets were vegetarian. In the case of the high- MUFA

diet, 13.0% dietary calories as carbohydrate was replaced by MUFA in the dietary

portfolio. MUFA was given in the form of a high-MUFA sunflower oil which contained

80% MUFA. Participants were also given the option to substitute a portion of the high-

MUFA oil with a calculated amount of avocado. The aim of the dietary portfolio was to

provide 1.0 g of plant sterols per 1000 kcal of diet in a plant sterol ester–enriched

margarine (Flora Pro-Activ, Unilever, London, England) with a minimum of 2g/d and a

maximum of 3g/d of plant sterols. The diet also provided 10.3 g of viscous fibres per

1000 kcal of diet from oats, barley, and psyllium; 20 g of soy protein per 1000 kcal as

soy milk, tofu and soy meat analogs; and 21.5 g of whole almonds per 1000 kcal of diet.

Emphasis was placed on eggplant and okra as additional sources of viscous fibre (0.2

g/1000 kcal and 0.4 g/1000 kcal, respectively). Eggs (2/wk) were also provided in the

dietary portfolio to balance the saturated fat and dietary cholesterol in the very low-

saturated-fat therapeutic control diet (Table 3). This dietary portfolio has been described

in detail previously (10).

The very low-saturated-fat therapeutic control diet as eaten in the first month

used skim milk, fat-free cheese and yogurt, and egg substitute and liquid egg white to

29

achieve a low intake of saturated fat. High fibre intake was obtained by the use of

wholegrain breakfast cereals (fibre, 2.5g/1000 kcal of diet) and bread (fibre, 3g/1000

kcal of diet) made from 100% whole wheat flour. This diet therefore lacked sources of

viscous fibres, plant sterols, soy protein and nuts. The macronutrient profile of the diet

recorded as consumed over the 4 weeks achieved a saturated fat intake of <5% of total

calories with <50mg cholesterol/1000kcal (Table 3).

Participants were provided with self-tarring electronic scales (Tanita

Corporations, Arlington Heights, USA) and asked to weigh all food items consumed

prior to and during the study period. During the study period, all foods to be consumed

by participants were provided initially by courier and then at weekly clinic visits, with the

exception of fruit and low calorie, non–starch-containing vegetables (8% and 24% of

dietary calories respectively from high- and low-MUFA diets). Okra was the exception

and was provided for the dietary portfolio. Participants were instructed to obtain specific

fruit and vegetables from their local stores and were reimbursed on presentation of

receipts. Participants were provided with 7-day rotating menus (Table 4). The diets have

been described in detail previously (10).

Dietary compliance was assessed from the completed weekly checklists and

from the return of uneaten food items.

4.3.4 Analyses

All serum samples from a given individual were labelled by code and analyzed in

the same batch. Serum was analyzed according to the Lipid Research Clinics protocol

(254) for total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C)

by detergent solubilization and measurement of HDL-C (Roche Hitachi 917; Roche

Diagnostics, Laval, Quebec, Canada), in the J. Alick Little Lipid Research Laboratory.

Low-density lipoprotein cholesterol (LDL-C) was calculated (255). Serum apoA1 and

apoB were measured by nephelometry, which is a measurement of scattered light by a

dilute suspension of small particles (Dade BehringBNProSpec; Dade Behring Canada

Inc, Mississauga, Ontario) (intra-assay coefficient of variation, 2.2% and 1.9%,

respectively) (256). Serum samples, stored at −70°C, were analyzed for hs-CRP by

end-point nephelometry (coefficient of variation, 3.5%) (Behring BN-100, N high-

30

sensitivity C-reactive protein reagent, Dade- Behring, Mississauga, Ontario). Diets were

analyzed using a program based on US Department of Agriculture data (257) and

developed in our laboratory to allow addition of data on foods relevant to ongoing

studies after analysis in the laboratory for protein, total fat and dietary fibre using

American Organization of Analytical Chemists methods and fatty acids by gas

chromatography (10).

4.3.5 Statistical Analysis Results were calculated as mean±SE. The study was an efficacy study with HDL-

C as the primary outcome in the direct comparison of high- and low-MUFA dietary

portfolio treatments. Since no within treatment difference was seen between weeks 2

and 4 and between weeks 6 and 8, the significance of the differences between

treatments was assessed by the CONTRAST statement in SAS using all post baseline

values (SAS PROC GLM) (258). The analysis used the change from baseline to weeks

2 and 4 for the very low-saturated-fat therapeutic control. The same weeks 2 and 4 of

the control diet, in turn, acted as the baseline for the high- and low-MUFA dietary

portfolios with weeks 6 and 8 as end of treatment response variables. Treatment, sex

and their interaction were main effects, with baseline (mean of control weeks 2 and 4 in

the portfolio analyses) as covariate. A 2-tailed paired t test was used to assess the

significance of the within treatment (weeks 2 and 4) percentage change from baseline

for the control and between weeks 2 and 4 (averaged) versus weeks 6 and 8

(averaged)for the high- and low-MUFA dietary portfolio groups. Means of weeks 2 and 4

data and weeks 6 and 8 data are presented in text and Tables.

With 12 participants per treatment group, and assuming an 8% SD of effect with

α=.05 and 1-β=.80, we had sufficient power to detect a 9.6% change in HDL-C between

treatments as significant.

4.4 RESULTS

4.4.1 Participants Twenty-four healthy, hyperlipidemic participants were randomized to either the

high- or low-MUFA arms of the study (17 men and 7 postmenopausal women) (Figure

31

1).Their baseline characteristics were similar (Table 1). Six participants had been taking

statins and, after obtaining approval from their primary care provider, discontinued them

as required by the study protocol at least 2 weeks prior to the study (3 low-MUFA and 3

high-MUFA dietary portfolio participants).

When expressed as the percentage of prescribed calories recorded as eaten

over the 4 week period, compliance was high for all 3 diets: 93±2% for the very low-

saturated-fat therapeutic control, 95±2% for the high-MUFA, and 91±2% for the low-

MUFA dietary portfolio. At the end of each treatment all participants believed they were

eating as much food as they were capable without experiencing discomfort (rating >3.0).

Satiety ratings for treatments were: control, 2.1±0.2; high-MUFA dietary portfolio,

2.3±0.3; and low-MUFA dietary portfolio, 2.0±0.3. Participants lost a similar amount of

weight on all 3 one- month treatments (control, -1.10±0.2kg; P<.001; high-MUFA dietary

portfolio, -0.78±0.21kg; P=.003; low-MUFA dietary portfolio, -0.98±0.26kg; P=.003).

4.4.2 Blood Lipids and C-Reactive Protein

For the stabilization control diet, no treatment differences in baseline or weeks 2

and 4 blood measurements were observed between the 2 treatment groups (Table 5)

who would subsequently be randomized to either high- or low-MUFA Portfolio diets

(Figures 2 and 3). The mean percentage change from baseline using the data for the 24

participants was -15.6±1.4% (P<.001) for total-C, -21.5±2.0% (P<.001) for LDL-C, and

7.9±2.7% (P=.008) for HDL-C. The percentage changes were also significant for the

total:HDL-C ratio (-21.9±1.9%, P<.001), apoA1 (3.7±1.6%, P=.028), apoB (-17.6±1.5%,

P<.001) and the apoB:A1 ratio (-20.4±1.9%, P<.001). However the change in TG was

not significant (-12.0±6.4%, P=.073).

For the dietary portfolio comparisons (Table 6), there were significant increases

on high-MUFA but not low-MUFA in HDL-C, 13.1±3.6% versus 2.7±3.6% (P=.002); and

apoA1, 7.8± 2.2% versus -0.4±1.5% (P=.001). There were also significant reductions on

high- versus low-MUFA in total:HDL-C, -24.5±2.1% versus -17.6±3.0% (P=.006); and

apoB:A1, -23.2±1.6% versus -17.6±2.3% (P=.013). Similarly, hs-CRP was reduced on

the high-MUFA, -37.5±10.6% but not on the low-MUFA dietary portfolio 45.3±33.4%

(P=.003) (Figures 2 and 3).

32

There were no between treatment differences in LDL-C or apoB. However,

significant within treatment reductions were seen on both high- and low-MUFA for LDL-

C, −20.9±2.6% (P<.001) versus −22.1±3.1% (P<.001) and apoB -17.2±2.1% (P<.001)

versus -18.0±2.2% (P<.001).

4.4.3 Blood Pressure

No significant treatment difference was observed in blood pressure (Tables 5 and 6).

4.4.4 Drop outs and Adverse Events

No participant withdrew from the study after the randomization which occurred

during the first and second week of the control phase. One participant withdrew at the

time of the first control breakfast, and therefore prior to randomization, due to the

perceived inconvenience of a metabolically controlled diet. No adverse events related to

the study protocol were reported.

4.5 DISCUSSION

The present study demonstrated that replacement of 13.0% of total calories from

carbohydrate by MUFA in the dietary portfolio, while not altering the LDL-C reduction,

resulted in a 10.4% greater increase in HDL-C over the 4 weeks. There was also a

6.9% greater reduction in the atherogenic index, total:HDL-C (247, 259) when

compared to the low-MUFA dietary portfolio. This increase in HDL-C is of similar

magnitude to two gemfibrozil studies which raised HDL-C concentrations by 6% and 8%

and resulted in reductions in the relative risk of CHD of 22% and 23%, respectively

(260, 261). The addition of MUFA by increasing HDL-C may therefore further enhance

the cardioprotective effect of the cholesterol-lowering dietary portfolio.

The lines of evidence for a cardioprotective effect for HDL-C include

epidemiologic and especially cohort studies (262-264). Measurements in the

Framingham cohort have resulted in inclusion of the total:HDL-C ratio as the lipid

parameter in the widely used Framingham CHD risk predictive equation (247) and post-

hoc analysis of statin trials have identified HDL-C as predictive of major cardiovascular

events even in participants whose LDL-C levels were optimized with a statin (243).

33

Intervention trials using gemfibrozil have also supported a cardioprotective role for

raising HDL-C (261, 265), as have studies of apoA1 infusion, the apolipoprotein of HDL,

which has been associated with plaque regression documented with intravascular

ultrasound measurements (88, 266). These data together with laboratory studies (267,

268) have supported the concept that HDL is involved in reverse cholesterol transport,

anti-oxidant and anti-inflammatory activities indicative of cardioprotective effects (97,

98).

Consequently, much emphasis has also been placed recently on lifestyle

strategies to raise HDL-C including exercise (16, 17), weight loss (16, 17), moderate

alcohol intake (16, 17), smoking cessation (16, 17), and increased monounsaturated

fatty acid intake (17, 99, 208, 269). The increased MUFA intake is the only strategy

relevant to the present study since participants were instructed to hold exercise

constant, weight loss was similar in all treatments, alcohol intake was very small (a

deviation from the dietary protocol but constant over both treatments), and no

participant smoked.

Although some reservations have been expressed over the use of

monounsaturated fats in preventing arteriosclerosis (270), previous studies have also

noted the advantages of monounsaturated fat on the blood lipid profile, especially in

type 2 diabetes, which is characterized by raised triglyceride and low HDL-C

concentrations (271). Early studies showed that glycemic control was improved, serum

triglycerides reduced and HDL-C increased with 25% substitution of carbohydrate by

MUFA (215). Later studies have shown more variable results using a 15% calorie switch

from carbohydrate to MUFA with reductions in VLDL-cholesterol and triglyceride but no

change in HDL-C (272-274). In the present study, triglyceride was significantly reduced

on the high- MUFA diet compared with the control diet but the treatment difference was

not significant. The higher HDL-C and apoA1 on the high-MUFA compared to the low-

MUFA diet resulted in significant reductions in the total:HDL-C and apoB:A1 ratios.

These changes would be expected to reduce the risk for CHD (247).

The mechanisms by which MUFAs increase HDL-C are likely to be several. They

include the ability of MUFA to scavenge free radicals (203, 275), reduce adipose tissue

synthesis of pro-inflammatory cytokines and the stimulus to hepatic acute phase protein

34

synthesis, such as CRP. This metabolic environment would also allow increased

synthesis of negative acute phase proteins, such as HDL, as seen in the present study.

Our data are consistent with such a hypothesis, since plasma CRP concentration was

reduced with the high-MUFA dietary portfolio. In addition, the displacement of dietary

carbohydrates by MUFA is likely to result in less carbohydrate induced hepatic VLDL-

TG synthesis(238). The resulting lower, VLDL-TG concentration, although not

significant in the present study, may attenuate the impact of cholesterol-ester transfer

protein (CETP) in depleting cholesterol ester of HDL (276, 277), thereby contributing to

a rise in plasma HDL-C concentration. The resulting TG-poor cholesterol-ester-rich HDL

would also be predicted to be less rapidly lost from the circulation by tissue uptake than

a TG-rich cholesterol-ester-poor particle (228, 238, 278) and this is consistent with the

observed increase in apoA1 concentrations after the high-MUFA dietary portfolio (99,

269).

The limitations of the study include the relatively small subject numbers, weight

loss, and the prescriptive nature of the diet, since adherence to the diet may be

considerably less when eaten as a self-selected diet under real-world conditions.

Despite these concerns, the effect size of the measurements of specific interest, HDL-C

and apoA1 and their ratios, were substantial. In our post-hoc analysis, although our

effect size for HDL-C was somewhat larger than expected (10.4% versus 9.6%), the

standard deviation of effect was also larger (12.5% versus 8%) and resulted in only 50%

power (α=0.05). Nevertheless, the apolipoprotein of HDL-C, apoA1, with an effect size

of 8.2% and a standard deviation of 6.6%, provided 83% power to detect the effect size

as significant in favour of MUFA (α=0.05). Even though there was weight loss on all

treatments, the loss of weight was similar and furthermore, did not influence the

outcomes when included as a covariate in the statistical model. In terms of metabolically

controlled conditions of the study, it is true that when the dietary portfolio has been

consumed under self-selected real-world conditions, a mean adherence to the

prescribed diet of only 64% was observed at 1 year (199). On the other hand, the high

degree of adherence (95 and 91%) recorded on the present metabolic diets (low- and

high-MUFA respectively) has allowed treatment differences to be more clearly

demonstrated with relatively small subject numbers.

35

It should also be emphasized that the participants were largely non-obese

(15/24), generally physically active, non-smokers on very low-saturated-fat diets. The

MUFA increase therefore represented an addition to an otherwise healthy lifestyle and

does not address the issue of the increased use of MUFA by those whose diet and

lifestyles may be suboptimal (270). It would therefore be of great interest to test how

addition of MUFA to an effective LDL-C lowering dietary portfolio can further increase

plasma HDL-C in individuals with low levels such as those with the metabolic syndrome

and type 2 diabetes.

The strengths of the study include the large effect size and the dietary metabolic

control that ensured the uniform and very specific exchange of carbohydrate for MUFA

in the diet. The study was also unique in determining the effect of adding MUFA to a

diet which already had powerful cholesterol-lowering ability due to the very low-

saturated-fat content and the use of nuts, viscous fibres (279), plant sterols (168, 171)

and soy products (126, 128). The present study therefore demonstrated that MUFA

preserved the HDL-C and apoA1 concentrations and so significantly increased the

therapeutic potential of this dietary approach (246, 260-262, 280).

The total reductions in LDL-C and the total:HDL-C ratio on both the high- and

low-MUFA dietary portfolio treatments which plateaued after 2 weeks were as large as

any reported in previous Portfolio studies (Figures 2 and 3) (10). Expressed as the

reduction from the start of the metabolic diets and including the reductions on both the

therapeutic control diet and the further decrease on the high- or low-MUFA dietary

portfolios, the total LDL-C reductions were 35.0% and 35.2% respectively. These LDL-C

reductions are similar to those recorded in many of the earlier statin trials which resulted

in 30-35% reductions in CHD risk (281).

At present, although there are no guidelines that specifically recommend raising

HDL-C, the NCEP ATP III have a cut point for HDL-C of ≥1.0mmol/L with cut points for

the metabolic syndrome of >1.0mmol/L for men and >1.3mmol/L for women (7) and the

2009 Canadian Cardiovascular Society Guidelines stratify cardiovascular risk for

therapeutic purposes based partially on total:HDL-C ratio of >4 for high, >5 for medium,

and >6 for low risk (18).

36

In conclusion, the CHD risk reduction potential of an effective cholesterol-

lowering diet may be significantly enhanced by inclusion of a moderate amount of

monounsaturated fat.

37

Figure 1: Patient flow diagram. *Chose not to participant (n=12): medical issue arose (n=4), want to lose weight (n=2), not willing to go off statin (n=1), not willing to stop vitamin supplements (n=1), not willing to give blood (n=1), not willing to do kinetics test (n=1), family issue (n=1), not interested (n=1).

Eligible after Telephone Screening (n=84)43M:41F

Excluded (n=59)59–Not meeting inclusion criteria

Drop Out (n=14)6–Chose not to participate*8-unable to contact

Excluded (n=1)1 –Not meeting inclusion criteria

Telephone screening (n=162)76M:86F

Attended Information Session (n=70)39M:31F

Drop Out (n=19)19 – time commitment

Drop Out (n=6)6-chose not to participate*

Clinical Assessment (n=63)34M:29F


Control Diet (n=25)17M:8F

Drop Out (n=27)27–scheduling difficulties

Drop Out (n=1)1-drop out after 1 day on diet

Randomized (n=24)17M:7F

Completion Control (n=24)17M:7F

Allocated to High-MUFA (n=12)8M:4F

12 Completed9M:3F

12 Completed8M:4F

Allocated to Low-MUFA (n=12)9M:3F

Eligible after Telephone Screening (n=84)43M:41F


Drop Out (n=14)6–Chose not to participate*8-unable to contact

Excluded (n=1)1 –Not meeting inclusion criteria

Telephone screening (n=162)76M:86F

Attended Information Session (n=70)39M:31F

Drop Out (n=19)19 – time commitment

Drop Out (n=6)6-chose not to participate*









12 Completed9M:3F

12 Completed8M:4F










12 Completed9M:3F

12 Completed8M:4F






12 Completed9M:3F

12 Completed8M:4F




12 Completed9M:3F

12 Completed8M:4F



12 Completed9M:3F

12 Completed8M:4F


38

Figure 2. Mean (SE) percentage change from baseline for both treatments for HDL-C,

high-density lipoprotein cholesterol (P=0.004); apoA1, apolipoprotein A1 (P=0.001);

total:HDL-C, total-cholesterol to high-density lipoprotein cholesterol ratio (P=0.045);

apoB:A1, apolipoprotein B to apolipoprotein A1 ratio (P=0.030) (significance of

difference between treatments) using the CONTRAST statement.

HDL-C

-35-30-25-20-15-10

-505

10

0 2 4 6 8

%C

hang

e B

asel

ine

Wk0

High-MUFA Low-MUFA

ApoA1

-35-30-25-20-15-10

-505

10

0 2 4 6 8Week

%C

hang

e B

asel

ine

Wk0

ApoB:A1 Ratio

-35-30-25-20-15-10

-505

10

0 2 4 6 8Week

%C

hang

e fro

m B

asel

ine

Wk0

Total:HDL-C Ratio

-35-30-25-20-15-10

-505

10

0 2 4 6 8

%C

hang

e fro

m B

asel

ine

Wk0

39

Figure 3. Mean (SE) percentage change from baseline for both treatments for total-C, total

cholesterol (P=0.517); LDL-C, low-density lipoprotein cholesterol (P=0.473); apoB,

apolipoprotein B (P=0.786); TG, triglyceride (P=0.538) (significance of difference between

treatments) using the CONTRAST statement.

Total-C

-40

-30

-20

-10

0

10

20

30

0 2 4 6 8

%C

hang

e fro

m B

asel

ine

Wk0 High-MUFA Low-MUFA

LDL-C

-40

-30

-20

-10

0

10

20

30

0 2 4 6 8Week

% C

hang

e fro

m B

asel

ine

Wk0

ApoB

-40

-30

-20

-10

0

10

20

30

0 2 4 6 8

TG

-40

-30

-20

-10

0

10

20

30

0 2 4 6 8Week

40

Mean ± SEM Range Mean ± SEM RangeAge (y) 55 ± 7 42 - 68 56 ± 11 38 - 69

Sex (female/male) 3/8 8/4

Weight (kg) 83 ± 4 62 - 113 80 ± 2 71 - 89

BMI (kg/m2) 29 ± 4 25 - 36 29 ± 2 26 - 32

Total-C (mmol/L) 6.3 ± 0.2 5.4 -7.7 6.2 ± 0.3 5.2 - 7.6

LDL-C (mmol/L) 4.3 ± 0.2 3.5 - 5.6 4.2 ± 0.2 3.3 - 5.2

HDL-C (mmol/L) 1.14 ± 0.08 0.73 - 1.66 1.22 ± 0.03 1.03 - 1.37

Triglycerides (mmol/L) 1.9 ± 0.2 1.0 - 3.5 1.6 ± 0.2 0.8 - 2.8

Systolic blood pressure (mmHg) 127 ± 3 109 - 142 121 ± 3 105 - 142

Diastlolic blood pressure (mmHg) 77 ± 3 57 - 91 74 ± 2 61 - 89

Exercise, METs 21.7 ± 5.2 1.5 - 55.0 23.2 ± 5.0 2.3 - 55.1

Medications, No. of subjects Blood Pressure 3 2 Thyroxine (0.08mg OD) 0 1 Aspirin 81mg 1 3 Supplements: Iron Supplementation (7mg TID) 7 9 Multivitamin 2 2 Calcium 3 5 Vitamin D 2 3 Glucosamine Chondritin 1 1Discontinued prior to study* Statins: n=3 n=3

Crestor 5mg 1Crestor 10mg 1 2Crestor 20mg 1Lipitor 20mg 1

Abbreviations: MUFA, monounsaturated fatty acid, BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; METs, metabolic equivalent of tasks (an index number expressing the energy cost of physical activities as multiples of Resting Metabolic Rate)SI conversion factors: To convert cholesterol and triglycerides to mg/dL, divide by 0.0259 and 0.0113, respectively.

† Data are expressed as mean±SE. No signficant baseline differences were observed between the high and low-MUFA groups.

Table 1. Baseline characteristics of randomized study participants.†High MUFA (n=12) Low MUFA (n=12)

* participants taking statins were asked to discontinue them 2 weeks prior to beginning the study

41

High MUFA Group(n=12)

Low MUFA Group (n=12)

Calories (kcal) 1882 1914Protein (%) 16.6 15.7Soy Protein (%) 0.0 0.6Available CH2O (%) 57.3 56.1Dietary Fiber (g/1000kcal) 18.6 17.5Viscous Fiber(g/1000kcal) 2.0 1.7Fat (%) 24.0 28.2SFA (%) 6.1 6.7MUFA (%) 9.6 11.5PUFA (%) 5.4 7.2Dietary Cholesterol (mg/1000kcal) 61.6 60.6Alcohol (%) 2.1 1.5

Table 2: Macronutrient Composition of the Participants' Habitual Diet* Habitual Diet:

Abbreviations: CH2O, carbohydrate; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid

*Data are expressed as mean percentage of calories per day unless otherwise noted.

42

Table 3: Macronutrient Composition of the Study Diets*NCEP Control Diet

(n=24)Low MUFA

Portfolio(n=12)

High MUFA Portfolio(n=12)

Calories (kcal) 2442a 2345a 2536a

Protein (%) 20.4a 21.7b 20.5a

Soy Protein (%) 0.0a 8.3b 7.7c

Available CH2O (%) 51.9a 49.1b 33.9c

Dietary Fiber (g/1000kcal) 20.1a 35.5b 32.7c

Viscous Fiber (g/1000kcal) 0.0a 11.0b 9.3c

Fat (%) 27.5a 29.3b 45.6c

SFA (%) 4.6a 4.7a 6.7b

MUFA (%) 10.6a 12.9b 25.9c

PUFA (%) 9.9a 11.0b 11.7b

Dietary Cholesterol (mg/1000kcal) 38.9a 29.9b 31.8c

Alcohol (%) 0.14a 0.00b 0.38c

Abbreviations: CH2O, carbohydrate; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; NCEP Control Diet, National Cholesterol Education Program-based Control Diet

*Data are expressed as mean percentage of calories per day unless otherwise noted where different letters as superscripts on the same line indicate significant differences between treatments (P<0.05) as assessed by ANCOVA.

43

Table 4: Example Menuplan of Diets for 2000kcal

Control Diet High MUFA Diet Low MUFA Dietgrams grams

Breakfast: Cereal: Hot Cereal: Hot Cereal: Bran flakes cereal* 30 Oat bran 40 Oat bran 40 Skim milk 220 Unsweetened Soy Beverage 250 Soy Beverage 200Fruit&Yogurt: Strawberries 50 Strawberries 150 Strawberries 100 Drink: Sugar 3 Fat Free Yogurt* 175 Psyllium (+ water 250ml) 7 Drink: Strawberry Jam* 10 Toast:: Psyllium (+ water 250ml) 7

Bread, Oatbran 65 Toast: Strawberry Jam* 15 Oatbran bread 30 Study margarine (Plant sterol-enriched) 12 Strawberry Jam* 12 High-MUFA Sunflower Oil 13 Study margarine (Plant sterol-enriched) 65

Snack Pear 133 Almonds 15 Almonds 15 Skim milk 220 Pear 102 Pear 197

Lunch Soup: Soup: Soup: Pasta & Fagioli Soup* 270 Barley Vegetable Soup* 45 Barley Vegetable Soup* 45Sandwich: Sandwich: Sandwich: Whole wheat bread 90 Oatbran bread 65 Oatbran bread 65 Light Margarine 35 Study margarine (Plant sterol-enriched) 13 Study margarine (Plant sterol-enriched) 13 Fat Free Cheese 42 Soy Deli Slices* 62 Soy Deli Slices* 62Salad: Salad: Salad: Lettuce, Romaine 30 Lettuce, Romaine 30 Lettuce, Romaine 30 Cucumber 50 Cucumber 50 Cucumber 50 Tomato 100 Tomato 100 Tomato 100 Oil, Olive 11 Almonds (chopped) 12 Almonds (chopped) 12 Vinegar 10 High-MUFA Sunflower Oil 12

Snack Ancient Grain Crackers* 10 Almonds 15 Almonds 15 Orange 133 Apple 103 Orange 197

Unsweetened Soy Beverage 250 Soy Beverage 200

Dinner Entre: Soup or stew: Soup or stew: Cheese Cannelloni* 246 Low-Fat Extra-Firm Tofu* 120 Low-Fat Extra-Firm Tofu* 120Bake/grill: Okra 100 Okra 100 Safflower Oil 15 Cauliflower 100 Cauliflower 100 Cauliflower 150 Onions 30 Onions 30 Onions 30 Green pepper 50 Green pepper 50 Green pepper 50 Pearled barley 35 Pearled barley 35 Fat Free Cheese 42 High-MUFA Sunflower Oil 18 Sunflower Oil 6

Snack Skim milk 220 Unsweetened Soy Beverage 225 Soy Beverage 150 Apple 133 Psyllium (+ water 250ml) 7 Psyllium (+ water 250ml) 7

Apple 196*these products were Blue Menu line products (Loblaws, Toronto, Canada)

44

Control prior to HighMUFA (n=12) Control prior to LowMUFA (n=12)Week 0 Week 2&4 Difference (SE) Week 0 Week 2&4 Difference (SE) P-value£

Body Weight, kg 83.1 (4.1) 82.4 (4.1) - 0.7 (0.3)* 79.9 (1.8) 78.9 (1.7) - 1.0 (0.3)† 0.556Cholesterol, mmol/L

Total-C 6.33 (0.20) 5.48 (0.20) - 0.85 (0.13)‡ 6.19 (0.26) 5.39 (0.26) - 0.80 (0.13)‡ 0.802LDL-C 4.34 (0.20) 3.67 (0.17) - 0.67 (0.11)‡ 4.24 (0.22) 3.53 (0.15) - 0.71 (0.12)‡ 0.803HDL-C 1.14 (0.08) 1.00 (0.05) - 0.14 (0.10)† 1.22 (0.03) 1.08 (0.03) - 0.14 (0.03)† 0.889

Triglycerides 1.87 (0.21) 1.77 (0.12) - 0.10 (0.17) 1.60 (0.17) 1.71 (0.34) 0.11 (0.26) 0.505Apolipoproteins, g/L

ApoA1 1.43 (0.05) 1.28 (0.05) - 0.14 (0.03)‡ 1.49 (0.04) 1.34 (0.05) - 0.15 (0.04)† 0.943ApoB 1.23 (0.05) 1.10 (0.04) - 0.13 (0.02)‡ 1.17 (0.06) 1.05 (0.05) - 0.12 (0.02)‡ 0.754

RatiosTotal:HDL-C 5.84 (0.40) 5.63 (0.29) - 0.21 (0.17) 5.1 (0.24) 5.03 (0.26) - 0.07 (0.18) 0.592LDL:HDL-C 4.01 (0.31) 3.78 (0.23) - 0.23 (0.10)* 3.50 (0.21) 3.30 (0.15) - 0.20 (0.12) 0.863

ApoB:A1 0.88 (0.05) 0.87 (0.05) - 0.01 (0.01) 0.80 (0.05) 0.80 (0.04) 0.00 (0.02) 0.883hs-CRP, mg/L 1.17 (0.25) 1.62 (0.24) 0.44 (0.25) 1.58 (0.42) 1.76 (0.59) 0.18 (0.24) 0.460Blood Pressure, mm Hg

Systolic 126.6 (3.0) 120.5 (2.4) - 6.1 (2.2)* 121.3 (2.8) 120.2 (2.4) - 1.08 (1.9) 0.100Diastolic 76.6 (2.8) 73.6 (2.3) - 3.0 (1.5) 73.5 (2.0) 70.9 (1.7) - 2.6 (1.7) 0.838

Table 5. Effect of Control, very low saturated fat diet, on Blood Lipids, C-Reactive Protein, and Blood Pressure in High- vs. Low-MUFA Groups

Abbreviations: MUFA, monounsaturated fatty acid; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; hs-CRP, high-sensitivity C-reactive protein* significantly different from Week 0 at P<0.05, † at P<0.01, ‡ at P<0.001;£ difference between changes in high- versus low-MUFA groups using the CONTRAST statement

To convert total-C, LDL-C, and HDL-C to mg/dL, divide by 0.0259; for triglycerides to mg/dL, divide by 0.0113; for ApoA1 and B to mg/dL, multiply by 100.

45

Week 2&4 Week 6&8 Difference (SE) Week 2&4 Week 6&8 Difference (SE) P-value£

Body Weight, kg 82.4 (4.1) 81.6 (4.0) - 0.78 (0.21)† 78.8 (1.7) 77.9 (1.6) - 0.98 (0.25)† 0.485Cholesterol, mmol/L

Total 5.48 (0.20) 4.66 (0.09) - 0.82 (0.13)‡ 5.39 (0.26) 4.51 (0.26) - 0.87 (0.11)‡ 0.517LDL-C 3.67 (0.17) 2.89 (0.13) - 0.78 (0.11)‡ 3.53 (0.15) 2.76 (0.18) - 0.77 (0.14)‡ 0.473HDL-C 1.00 (0.05) 1.13 (0.07) 0.13 (0.03)† 1.08 (0.03) 1.11 (0.05) 0.03 (0.05) 0.004

Triglycerides 1.77 (0.12) 1.41 (0.09) - 0.36 (0.07)‡ 1.71 (0.34) 1.43 (0.16) - 0.28 (0.34) 0.538Apolipoproteins, g/L

A1 1.28 (0.05) 1.38 (0.06) 0.10 (0.03)† 1.34 (0.05) 1.33 (0.05) - 0.01 (0.02) 0.001B 1.10 (0.04) 0.90 (0.03) - 0.19 (0.03)‡ 1.05 (0.05) 0.87 (0.05) - 0.19 (0.03)‡ 0.786

RatiosTotal:HDL-C 5.63 (0.29) 4.21 (0.18) - 1.42 (0.17)‡ 5.03 (0.26) 4.12 (0.21) - 0.91 (0.22)‡ 0.045LDL:HDL-C 3.78 (0.23) 2.62 (0.14) - 1.16 (0.12)‡ 3.30 (0.15) 2.52 (0.16) - 0.78 (0.10)‡ 0.040

ApoB:A1 0.87 (0.05) 0.67 (0.03) - 0.21 (0.02)‡ 0.80 (0.04) 0.65 (0.03) - 0.14 (0.02)‡ 0.030hs-CRP, mg/L 1.62 (0.24) 0.82 (0.13) - 0.79 (0.21)† 1.77 (0.59) 1.85 (0.55) 0.22 (0.46) 0.011Blood Pressure, mm Hg

Systolic 120.6 (2.4) 119.6 (2.1) - 1.00 (2.52) 120.3 (2.4) 117.3 (2.4) - 2.96 (1.74) 0.265Diastolic 73.5 (2.3) 72.5 (2.2) - 0.96 (1.36) 71.0 (1.7) 70.1 (1.7) - 0.88 (1.51) 0.428

* significantly different from Week 2&4 at P<0.05, † at P<0.01, ‡ at P<0.001; £ difference between changes in high- versus low-MUFA groups using the CONTRAST statementTo convert total-C, LDL-C, and HDL-C to mg/dL, divide by 0.0259; for triglycerides to mg/dL, divide by 0.0113; for ApoA1 and B to mg/dL, multiply by 100.

Table 6. Effect of High-MUFA and Low-MUFA Dietary Portfolio Treatments on Blood Lipids, C-Reactive Protein, and Blood PressureLow MUFA Portfolio (n=12)High MUFA Portfolio (n=12)

Abbreviations: MUFA, monounsaturated fatty acid; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; hs-CRP, high-sensitivity C-reactive protein

46

5. The Effect on Oxidative Stress of Adding Monounsaturated Fat to a Dietary Portfolio of

Cholesterol-Lowering Foods

47

The Effect on Oxidative Stress of Adding Monounsaturated Fat to a Dietary Portfolio of Cholesterol-Lowering Foods

5.1 Abstract

Background: Higher monounsaturated fat (MUFA) intakes may reduce oxidative damage

and contribute to reduced cardiovascular disease (CVD) risk by decreased oxidative

damage.

Methods: Twenty-four hyperlipidemic subjects took a very low-saturated-fat therapeutic

control diet for one month and then either a high- or low-MUFA dietary portfolio for a

further month. Food was provided for the 2-month period based on Harris-Benedict

estimates for requirement. Oxidative stress was measured as serum protein thiols and

conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) in the LDL

fraction.

Results: Although all treatments showed antioxidant activity, there were no significant

differences between the high- and low-MUFA dietary portfolios in any of the measures of

oxidative damage.. For the group as a whole (24 subjects), during the control diet there

were significant increases in protein thiols of 7.6 ± 3.1% (P=0.023) and significant

reductions in CD of 9.3 ± 3.0% (P=0.005) but non-significant reductions in TBARS of 8.5 ±

5.8% (P=0.154). During both the high- and low-MUFA dietary portfolios, protein thiols

increased significantly by 8.8 ± 3.7% (P=0.026) while CD and TBARS decreased

significantly by 9.6 ± 3.5% (P=0.012) and 8.5 ± 3.5% (P=0.021) respectively, indicating a

reduction in oxidative damage.

Conclusions: No effect of MUFA could be detected, although all diets showed antioxidant

activity.

48

5.2 INTRODUCTION

Evidence suggests that dyslipidemia and oxidative stress are key mechanisms

associated with the development of atherosclerosis and cardiovascular disease (CVD)

(282). Dyslipidemia has been closely associated with increased endothelial production

of reactive oxygen species (ROS) and the oxidative modification of low-density

lipoprotein (LDL)-associated lipids which is directly involved in the initiation of

atherosclerotic development (282). The dietary portfolio appears to reduce CVD risk

through reduction of both LDL-C and C-reactive Protein (CRP) (10, 11). Oxidized LDL

(ox-LDL), a further risk factor, may also be reduced because of the nature of the key

components of the dietary portfolio (12). Reductions in ox-LDL have been demonstrated

in studies involving soy protein or almonds (114, 115, 119, 120, 145, 146) and in some

studies on plant sterols and fibres, although the data are not as strong (165, 184, 185,

187). It was therefore of interest to determine whether the addition of monounsaturated

fatty acids (MUFAs) to the dietary portfolio provided a further benefit in terms of

antioxidant since MUFA consumption has been shown to reduce oxidative modification

of LDL (203, 240, 283).

We have therefore compared the effect on oxidative stress of substituting 13.0%

of total calories as MUFA for carbohydrate in an effective cholesterol-lowering dietary

portfolio.

5.3 METHODS

5.3.1 Participants

Men and post-menopausal women with mild to moderate hypercholesterolemia

were recruited and completed the study (17 men and 7 postmenopausal women). Their

mean ± SE age was 55 ± 9 years (range, 38-69 years) and mean body mass index

(BMI) was 28.8 ± 0.7 kg/m2 (range, 24.4-36.0 kg/m2) (refer to Table 1 in Chapter 4). All

participants had previously demonstrated high LDL-C levels >4.1 mmol/L (19, 242). No

participants had a history of cardiovascular disease, untreated hypertension (blood

pressure >140/90mmHg), diabetes, or renal or liver disease, and none were taking

medications known to influence serum lipids apart from 1 women taking stable doses of

thyroxine. Six participants were taking statins and had discontinued them as required at

49

least 2 weeks prior to the study (3 low-MUFA and 3 high-MUFA dietary portfolio

participants). All medications and supplements were expected to be taken at a constant

dose prior to and during the study. Five participants were taking antihypertensive

medications at a constant dose prior to and during the study and 4 were taking aspirin

or other anti-inflammatory drugs. Other, more commonly used non-prescription drugs

and supplements taken throughout the study period included a daily multivitamin (n = 4),

calcium (n = 8), glucosamine (n=2), melatonin (n=2) and vitamin D (n=5). Iron

supplementation (ferrous gluconate 7mg tid) was provided to 16 subjects on the basis of

a prestudy ferritin of <50mcg/L.

5.3.2 Study Protocol The study followed a randomized parallel design and was carried out between

August 2007 and April 2009. Participants followed their own low-saturated-fat

therapeutic diet for 1 month prior to the start of the study. They were then placed on a

very low-saturated-fat dairy and whole-grain cereal diet which acted as a stabilization

period prior to the high- and low-MUFA diets. After one month on this metabolically-

controlled diet, they were randomized to either a high-MUFA or conventional (low-

MUFA) dietary portfolio for a final month. All foods were provided except for fresh fruit

and vegetables which subjects were prescribed and instructed to obtain for themselves.

Throughout the study participants attended weekly clinic visits where body weight

was taken and blood pressure was measured three times in the non-dominant arm

using a mercury sphygmomanometer by the same observer. At 2-week intervals blood

samples were obtained after 12-hour overnight fasts. A seven-day diet history was

obtained for the week prior to the 2-month metabolic treatment period. Completed menu

checklists were returned at weekly intervals during the 4-week diet periods and checked

by the dietitians, who also recorded the participants’ previous week’s exercise and

ensured that it was constant over the course of the study period. At weekly intervals,

participants recorded their overall feeling of satiety using a 9 point bipolar semantic

scale in which −4 was extremely hungry, 0 was neutral, and +4 was uncomfortably

satiated.

50

The statistician stratified the participants on the basis of sex and an LDL-C of

4.2mmol/L in the first 2 weeks of the very low-saturated-fat therapeutic control diet using

a random number generator and SAS version 9.1 software (SAS Institute Inc, Cary, NC)

in a separate location from the clinic. The dietitians were not blinded to the diet because

they were responsible for patients’ diets and for checking diet records. The laboratory

staff responsible for analyses were blinded to treatment and received samples labelled

with name codes and dates. The study was approved by the ethics committees of the

University of Toronto and St. Michael’s Hospital and the Natural Health Products

Directorate of Health Canada. Written informed consent was obtained from all

participants. The clinical trial registration number is NCT00430430.

5.3.3 Diets

The diets eaten before the 8-week study were the participants’ routine

therapeutic low-fat diets, which were similar to current National Cholesterol Education

program guidelines (<7% energy from saturated fat and <200 mg/d of dietary

cholesterol) (19, 284) and previously referred to as a Step II diet (248). During the 8-

week study period, diets were provided based on estimated caloric requirements by the

Harris-Benedict equation and all diets were vegetarian. In the case of the high-MUFA

diet, 14.7% dietary calories as starch was replaced by MUFA in the dietary portfolio.

The aim of the dietary portfolio was the same as previous dietary portfolio studies (10,

198): to provide 1.0 g of plant sterols per 1000 kcal of diet in a plant sterol ester–

enriched margarine with a minimum of 2.0g and a maximum of 3.0g of plant sterols per

day; 10.3 g of viscous fibres per 1000 kcal of diet from oats, barley, and psyllium; 20 g

of soy protein per 1000 kcal as soy milk and soy meat analogs; and 21.5 g of whole

almonds per 1000 kcal of diet. Eggs (2/wk) were provided in the dietary portfolio to

balance the saturated fat and dietary cholesterol in the very low-saturated-fat

therapeutic control diet (refer to Table 4 in Chapter 4). The very low-saturated-fat

therapeutic control diet has been previously described (refer to Chapter 4). The

macronutrient profile of the diet recorded as consumed over the 4 weeks achieved a

saturated fat intake of <5% of total calories with <50mg cholesterol/1000kcal.

51

Participants were provided with self-tarring electronic scales (Tanita

Corporations, Arlington Heights, USA) and asked to weigh all food items consumed

prior to and during the study period. During the study period, all foods to be consumed

by participants were provided initially by courier and then at weekly clinic visits, with the

exception of fruit and low-calorie, non–starch-containing vegetables (8% and 24% of

dietary calories respectively from high- and low-MUFA diets). Okra was the exception

and was provided in the dietary portfolio. Participants were provided with 7-day rotating

menus (refer to Table 3 in Chapter 4).

Compliance was assessed from the completed weekly checklists and from the

return of uneaten food items.

5.3.4 Analyses Serum lipids were measured on samples stores at -70°C in the same batch

according to the Lipid Research Clinics protocol (254) for total-C, TG, and HDL-C after

dextran sulphate–magnesium chloride precipitation (285) and LDL-C was calculated

(255). Serum apolipoprotein A1 and B were measured by nephelometry (intra-assay

coefficient of variation, 2.2% and 1.9%, respectively) (256) and hs-CRP by end-point

nephelometry (coefficient of variation, 3.5%) (Behring BN-100, N high-sensitivity C-

reactive protein reagent, Dade- Behring, Mississauga, Ontario). All samples from a given individual were labelled by code and analyzed in the

same batch. Protein oxidation was measured using the 5,5′-dithiobis (2-nitrobenzoic

acid) assay (38) to assess the loss of reduced thiol (−SH) groups as a measure of

oxidation. The coefficient of variation of serum samples analyzed in triplicate was 3.0%.

LDL oxidation was estimated by measuring in the LDL fraction both CD, based on the

method of Ahotupa et al (39), and TBARS, using the malondialdehyde (MDA)–

thiobarbituric acid assay adopted from Jentzsch et al (40). The coefficients of variation

for these analyses were 5.7% and 6.4% for TBARS and CD, respectively.

Diets were analyzed using a program based on US Department of Agriculture

data and developed in our laboratory to allow addition of data on foods relevant to

ongoing studies after analysis in the laboratory for protein, total fat, and dietary fibre

52

using American Organization of Analytical Chemists methods and fatty acids by gas

chromatography (10).

5.3.5 Statistical Analysis Results were calculated as mean±SE. The significance of the differences

between the treatments for the oxidative markers were assessed using ANOVA in SAS

(258) with the change from baseline to week 4 for the very low-saturated-fat therapeutic

control diet and from week 4 to week 8 for the high- and low-MUFA dietary portfolios.

Treatment and sex and their interaction were main effects, with baseline as covariate. A

2-tailed paired t test was used to assess the significance of the within treatment (weeks

0 and 4) percentage change from baseline for the control and between week 0 versus

week 8 for the high- and low-MUFA dietary portfolio groups.

The original power calculation for the study was based on the ability to detect

changes in lipids and lipoproteins (refer to Chapter 4). In the present analysis, with the

observed SD of effect of 16.9% for TBARS and 12 subjects per group, we could be able

to detect a 20.2% difference in the primary measure (α=0.05, 1- β=0.8); for CD , with

the observed SD of effect of 17.6% for CD, we could be able to detect a 21.0%

difference in CD between the 2 treatments; and similarly for protein thiols, with the

observed SD of effect of 18.5%, we could be able to detect a 22.1% difference.

5.4 RESULTS 5.4.1 Participants

No significant differences were found in baseline data between the 2 groups,

both diets were similarly well complied with (>90%), there was no difference between

satiety ratings between the 2 groups, and participants lost a similar amount of weight on

all 3 one- month treatments (control, -1.10 ± 0.2kg; P<.001; high-MUFA dietary portfolio,

-0.78 ± 0.21kg; P=.003; low-MUFA dietary portfolio, -0.98 ± 0.26kg; P=.003).

5.4.2 Markers of Oxidation No differences were observed among the 2 treatment groups in baseline

oxidative measurements and no differences were seen between the high- and low-

53

MUFA diets. Increases in protein thiols and reductions in CD and TBARS were

observed throughout the study on both high- and low-MUFA diets (Tables 1 and 2). The

mean percentage change over the control month using the data for the 24 subjects for

protein thiols was 7.6 ± 3.1% (P=0.023) and for CD and TBARS was 9.3 ± 3.0%

(P=0.005) and 8.5 ± 5.8% (P=0.154), respectively. For the 24 subjects during the

portfolio diets, protein thiols further increased significantly by 8.8 ± 3.7% (P=0.026)

while CD and TBARS further decreased significantly by 9.6 ± 3.5% (P=0.012) and 8.5 ±

3.5% (P=0.021) respectively, indicating a reduction in oxidative damage (Figure 1). In

post-hoc analyses, there were no differences in oxidative measures between subjects

when they were divided into those receiving iron supplementation or those who lost

weight (data not shown).

5.4.3 Drop out and Adverse Events

No participant withdrew from the study after the randomization which occurred

during the first and second week of the control phase. One participant withdrew at the

time of the first control breakfast due to the perceived inconvenience of a metabolically

controlled diet. No adverse events were reported.

5.5 DISCUSSION This study demonstrated that replacement of 13.0% carbohydrate by MUFA

resulted in no differences between treatments for the oxidative markers protein thiols,

CD or TBARS. However, the antioxidant activity of the very low-saturated-fat

therapeutic control diet and both the high- and low-MUFA dietary portfolios were

reflected in the progressive increase in protein thiols and reduction in conjugated dienes

and TBARS and therefore present a potential reduction in LDL atherogenicity (22, 30,

31).

In the present study, the very low-saturated-fat control diet can be seen as a

positive control since low-fat dairy diets emphasizing fruits and vegetables, such as in

the Dietary Approaches to Stop Hypertension (DASH) trial, have previously

demonstrated reductions in markers of oxidation, as well as improved serum lipid

profiles (50). The continued improvement in the oxidative measures during the second

54

month of the study in which the subjects were consuming the dietary portfolio is

anticipated since many of the key components, including plant sterols, soy protein,

viscous fibres and almonds, have previously demonstrated the potential to reduce

oxidative stress such as reductions in ox-LDL (119, 145, 146, 183, 185), thus

suggesting the potential to protect LDL from oxidative damage (147, 148). Additionally,

almonds have demonstrated increases in total antioxidant capacity (120) and their skins

have shown antioxidant activity against free radicals in humans (121, 122) while whole

grains have been associated with reductions in risk of inflammatory diseases (166).

Therefore the dietary portfolio contains a variety of foods which may explain the

observed reduction in oxidative markers.

Although studies adding MUFA to a diet have demonstrated a reduction in

oxidative damage, such as reductions in the oxidative modification of LDL particles

(203), no difference between the high- and low-MUFA treatments were observed in the

present study. However, in the low-MUFA diet, MUFA was replaced by carbohydrates

which came from fruits, which resulted in a diet containing around 24% of its dietary

calories from fruit and vegetable as opposed to the 8% in the high-MUFA diet.

Therefore, since MUFA was replaced by fruits which are known to be rich in

antioxidants, it may be that there was no significant difference between the high- and

low-MUFA diets because both diets were rich in antioxidant foods of a different nature.

For example, some studies have demonstrated an approximate 10-15% reduction in

CDs on a high-MUFA versus low-MUFA diet over 1 month, (203, 286), while the DASH

(Dietary Approaches to Stop Hypertension) diet which contained about 16% of calories

from fruits and vegetables demonstrated a 10% reduction in urinary isoprostanes, a

marker of oxidative damage. Therefore, whether from increased MUFA or from a diet

rich in fruits and vegetables, there were similar reductions in markers of oxidative

damage. These reductions are similar to those observed in the present study where

there were reductions of 8.5% in CDs on the DASH-like control diet and another 10%

during the high-MUFA dietary portfolio. The further 10% reduction in CDs on the low-

MUFA dietary portfolio may be the result of the added fruit as well as the dietary

portfolio components.

55

Additionally, due to the inclusion of multiple foods with antioxidant potential

including a healthy DASH-like control diet, MUFAs, and fruit and vegetables, a

possible reason as to why no additional improvement in oxidative markers resulted from

the high-MUFA treatment is that the dietary benefit may have already been maximal

since some ability to mount an oxidative response is required for normal functioning

including defence by white cells from invading bacteria (287).

However, it is also possible that we did not see treatment differences in the

oxidative measures because of the relatively small sample size of 12.

The present study demonstrates that therapeutic diets rich in fruit, vegetables, soy,

nuts and other antioxidant components result in a reduction in oxidative damage. No

treatment difference was seen when MUFA was included in the diet possibly due to the

displacement of other antioxidant dietary components. Furthermore, because of the

high level of natural antioxidants, no effect of iron status was observed in this relatively

small group of subjects. More extreme situations in which there is a lack of antioxidant

activity provided by the control diet may have to be employed to demonstrate the effect

of dietary antioxidants.

56

Protein Thiols by Treatment

200

250

300

350

400

450

500

550

-2 0 2 4 6 8

Week

Prot

ein

Thio

ls (u

M)

Absolute CD by Treatment

20

30

40

50

60

70

80

90

100

-2 0 2 4 6 8

Week

Abso

lute

Con

juga

ted

Dien

es (u

M)

Absolute TBARS by Treatment

0.0

0.5

1.0

1.5

2.0

-2 0 2 4 6 8

Week

Abso

lute

TBA

RS (u

M)

Figure 1. Means of oxidative stress on high [n=12, ] and low [n=12, ] MUFA diets. 1 outlier was removed from the high-MUFA group in the analysis of CD (127.2, 102.3, 125.0 at Week 0, 4, 8, resp.). 1 outlier was removed from the low-MUFA group in the analysis of protein thiols (200.4 at week 8) and of CD (95.6 at week 8) and another outlier in the analysis of TBARS (2.7, 1.5, 1.4 at week 0, 4, 8, resp.).

57

High vs. Low Group

Week 0 Week 4 Difference (SE) Week 0 Week 4 Difference (SE) P-value

Protein Thiols (mmol/L) 328 ± 21 347 ± 20 20 ± 12 342 ± 15 369 ± 24 27 ± 16 0.261

Conjugated Dienes

Absolute Concentration (umol) 65 ± 6 60 ± 6.4 - 5 ± 3 66 ± 7 57 ± 6 - 8 ± 3* 0.499

Molar ratio of LDL-C (umol/mmol) 15.0 ± 1.5 16.0 ± 1.7 1.0 ± 0.8 14.3 ± 1.2 14.7 ± 1.0 0.4 ± 0.5 0.514

TBARS

Absolute Concentration (umol) 1.28 ± 0.15 1.03 ± 0.07 - 0.25 ± 0.10* 1.16 ± 0.11 1.05 ± 0.09 - 0.11 ± 0.08 0.284

Molar ratio of LDL-C (umol/mmol) 0.29 ± 0.04 0.27 ± 0.02 - 0.02 ± 0.02 0.26 ± 0.02 0.28 ± 0.03 0.02 ± 003 0.330

* significantly different from Week 0 (P<.05)

Table 1. Effect of Control, very low-saturated-fat diet, on Oxidative Markers in High vs. Low Groups

Control-High-MUFA (n=12)Control-Low-MUFA (n=12)

High vs. Low

Week 4 Week 8 Difference (SE) Week 4 Week 8 Difference (SE) P-value

Oxidative Markers:

Protein Thiols (mmol/L) 347 ± 20 376 ± 23 29 ± 15 369 ± 24 391 ± 18 22 ± 21 0.555

Conjugated Dienes

Absolute Concentration (umol) 60 ± 6 53 ± 5 - 7 ± 4 57.± 6 52 ± 7 - 5 ± 4 0.669

Molar ratio of LDL-C (umol/mmol) 16.0 ± 1.7 17.5 ± 1.6 1.6 ± 1.0 14.7 ± 1.0 17.0 ± 1.9 2.3 ± 1.2 0.651

TBARS

Absolute Concentration (umol) 1.03 ± 0.07 0.96 ± 0.06 - 0.07 ± 0.05 1.05 ± 0.09 0.89 ± 0.05 - 0.16 ± 0.06* 0.221

Molar ratio of LDL-C (umol/mmol) 0.27 ± 0.02 0.32 ± 0.02 0.05 ± 0.02* 0.28 ± 0.03 0.30 ± 0.03 0.02 ± 0.01 0.254

* significantly different from Week 4 (P<.05)

Table 2. Effect of Low-MUFA and High-MUFA Dietary Portfolio Treatments on Oxidative Markers

Low MUFA Portfolio (n=12) High MUFA Portfolio (n=12)

58

6. Overall Discussion and Limitations

59

6.1 Overall Discussion Dietary modification has been shown to reduce risk of CVD (19, 269) which

explains the continuous search for better dietary strategies to target multiple risk factors.

Just as 4 foods that had been individually shown to reduce cholesterol demonstrated an

additive effect when combined to form the dietary portfolio, MUFA was added in an effort

to further reduce the overall risk for CVD. As recent studies demonstrate, there is a

residual risk that remains after effective LDL-C reduction. Therefore, strategies to target

this residual risk are of great interest.

In this study, the high-MUFA treatment was able to effectively raise HDL-C

significantly more than the low-MUFA treatment. This is an important result since studies

indicate that there is a 1-3% associated reduction in risk of CVD for every 0.026mmol/L

increase in HDL-C (53-55, 57). Additionally, the lipid results of the high-MUFA treatment

demonstrate that not only is HDL-C being improved, but also apoA1 levels, which may

be an important indication of the quality of HDL since apoA1 has important roles

associated with HDL-C. For example, in RCT apoA1 is the ligand for the ABC (ATP-

binding cassette) transporter, and therefore is the docking point for which HDL can

receive cholesterol from peripheral cells and transport it to the liver (96). It is also the

cofactor for LCAT (lecithin cholesterol acyltransferase) which is important in removing

cholesterol from tissues and transporting them via HDL to the liver (96). Also, this study

demonstrates that in addition to the significant reduction in LDL-C, one of the possible

factors contributing to residual CVD risk, HDL-C, was also improved thereby potentially

providing further CVD risk reduction, especially for those who ~20% of people who attain

low LDL-C but who may still experience a cardiovascular event (53, 288).

The significant increase in HDL-C is also an important finding of the study

because it provides strong support for the idea that MUFA consumption can raise HDL-

C, which has been largely debated. The present study provides strong evidence because

of the following: it included a 4-week metabolically controlled run-in period (the low-

saturated-fat control diet) which acted as a stabilization period for all subjects prior to the

randomization to high- or low-MUFA portfolio diets; it was very well controlled, with all

foods provided to the participants’ homes, as well as reimbursement for any items

purchased for the study diet, there were weekly clinic visits for counselling with

60

suggestions for new recipes and ideas on how to combine foods for each participants’

personal preferences, as well as to assist in any difficulties encountered; and it provided

a moderate but significant amount of MUFA in the form of an enriched sunflower oil

which could easily be consumed in daily food preparation. Therefore, it allowed for all

subjects to attain comparable baseline values as a result of the stabilization period,

provided the tools for successful adherence to the diets, and achieved a significant

difference between treatments making them suitable for comparison.

The significant HDL-C increase on the high-MUFA diet is also promising because

the other methods of raising HDL-C, such as through the use of fibrates or niacin

therapy, have negative side effects making them undesirable. Niacin has been

associated with flushing, dry skin, and gastrointestinal irritation, as well as increased risk

of hepatotoxicity when taken as a monotherapy (17). Fibrates are less effective

compared to niacin therapy in raising HDL-C, and may also increase serum creatinine

and homocysteine levels and thus require close patient follow-up (17). Therefore, the

results of this study are very appealing because the high-MUFA was able to raise HDL-C

to a comparable level to that of the mentioned pharmacotherapies, allowing for a

convenient and effective substitution in the diet without the undesired side effects.

The other beneficial observation in the present study which may provide further

CVD risk reduction was the significant reduction in hs-CRP in the high-MUFA group

compared to the low-MUFA group. Thus, added benefit may arise from the anti-

inflammatory properties of HDL, or the result of apoA1 since evidence exists that the

interaction between apoA1 and the ABC transporter can act as an anti-inflammatory

receptor in a separate pathway from that in RCT (289). CRP is receiving greater

attention due to the recently published JUPITER trial which demonstrated the lowest

cardiovascular event rate was achieved in subjects with an LDL-C level less than

2.0mmol/L and an hs-CRP level less than 2.0mg/L (290). As a result of this trial, hs-CRP

has been incorporated into recommended clinical guidelines, such as in the Canadian

Cardiovascular Society Guidelines in which it is stated that in intermediate-risk groups

with LDL-C levels which do not require treatment, hs-CRP is part of risk stratification in

addition to its inclusion as a secondary target (18). Additionally, the total-HDL-C ratio is

in the guidelines as a secondary target and is included as a specific therapy target in

61

those classified as intermediate risk (18). Many studies have also concluded that it is one

of the best predictors of long-term risk (17, 18, 291, 292). In the present study, both hs-

CRP and total:HDL-C ratio were significantly reduced in the high-MUFA group, thus

there is the potential for MUFA to assist in improving these targets for CVD risk

reduction. Some more recent studies suggest that the apoB:A1 ratio may be a better

predictor of CVD risk than total:HDL-C ratio, however in the present study, the apoB:A1

ratio was also significantly reduced on the high-MUFA diet versus the low-MUFA diet,

therefore the same dietary interventions would be appropriate to help reduce risk via this

alternative ratio predictor (81).

Since LDL-C has a well established direct relationship with the development of

atherosclerosis and its oxidation appears to accelerate this process, strategies to reduce

oxidative stress are recognized as important (22, 30). Although no treatment differences

were observed in markers of oxidative stress, the diets as a whole were antioxidant and

may assist in CVD risk reduction. Firstly, both dietary portfolios reduced LDL-C by 20-

22%, which was preceded by a 15.5% reduction on the stabilization control diet,

therefore reducing the substrate itself in the atherosclerotic development process. Over

the 2 month period, LDL-C was reduced by about 1.5mmol/L in both treatment groups

which when applied to previously established and well recognized findings that

1.0mmol/L reduction in LDL-C is associated with a 21% reduction in CVD risk (20), these

diets would be associated with a 31.5% reduction in risk of CVD. Secondly, irrespective

of MUFA intake, there was a significant increase in protein thiols and significant

reductions in CD and TBARS, which overall indicate reductions in oxidative damage both

to proteins and lipids. Therefore, diets which are rich in whole grains and low-fat dairy, as

in the control diet, may reduce oxidative damage and diets which also contain soy, nuts,

and are rich in fruits and vegetables or MUFAs, as in the dietary portfolio treatments,

may provide further reductions. Although there is controversy over the effects of

antioxidants on oxidative damage, the majority of studies are looking at supplementation

in which case you may not get the appropriate diversity of antioxidants required to

observe expected benefits. In the present study, the control diet provided reductions due

to a few foods with some potential effects which was followed by greater reductions seen

on the dietary portfolio containing additional foods with potential benefit in addition to

62

either more fruit or high MUFAs. Thus, by providing an increment in variety of foods with

antioxidant potential, the study was able to demonstrate continuous reductions in

oxidative damage. Therefore, by using whole foods, there is a wider variety of

components which may act together to elicit effects on oxidative damage and thus may

contribute to reduction in CVD risk (293).

Overall, through the combination of foods which lower cholesterol and oxidative

damage, including the use of MUFAs, there is potential for greater reductions in CVD

risk.

6.2 Limitations and Future Directions In considering the limitations of the study, the prescriptive nature of the diet must

be addressed since the subjects received all their foods by courier and were given

menuplans indicating all foods to be eaten and at what amounts. The dietary portfolio

has been observed under real-world conditions with an adherence rate of 64% at 1-year.

However, with the suggested inclusion of MUFA, MUFA must be encouraged not to

displace the portfolio components but to replace other sources of fat in the diet and not to

be incorporated as an addition to the existing diet, which could easily result in weight

gain.

Another limitation was the weight loss which occurred throughout the duration of

the study period, as well as the relatively small sample size (n=24). The weight loss may

have been the result of the use of the Harris-Benedict predictive equation for energy

requirement which had quite a large activity factor that the present group of subjects

generally fell just under, so may not have been prescribed amounts that accounted for

their exercises. Also, they may not have actually been consuming the entire amount

prescribed because of remaining amounts in the cooking pans, in transferring, or leftover

on their plates. However, weight loss was consistent throughout the study and similar

among both diet groups and did not influence any study outcomes when included as a

covariate in the statistical model. In terms of our sample size, although small, we had

enough power to detect the effect size in apoA1 as significant in favour of MUFA

(α=0.05).

63

We may have been unable to detect differences in the oxidative markers between

the high- and low-MUFA groups because the nature of the foods which MUFA replaced

have been shown to have antioxidant activity, that is, fruit. Ideally, to be able to observe

the true effects of MUFA on oxidative damage, the reference or control diet would have

had little to no antioxidant effect. However, we wanted to compare the effect of adding

MUFA to a dietary portfolio, which already contains potential antioxidant activity in its

components. Also, in order to keep all the portfolio components at their prescribed levels,

the only carbohydrate source which would be available for replacement was that from

fruit. Therefore the reference diet, low-MUFA, was rich in foods also capable of

antioxidant activity, albeit with an unknown comparability to the activity of MUFAs.

6.3 Future Research Since there was no difference between the high- and low-MUFA dietary portfolios

in the reduction in oxidative damage demonstrated in this study, it would be interesting to

test other measures of oxidative damage given that similar studies have found

differences although the carbohydrates replaced in those studies contained more simple

sugars and low- fat dairy (203). This could include other measures of lipid oxidation, such

as isoprostanes, and protein oxidation, such as protein carbonyls, as well as measures

of oxidative damage to DNA, such as 8-hydroxydeoxyguanosine (8-OHdG) (294). It may

also be interesting to test if the samples from the high-MUFA groups had greater

antioxidant activity by such tests as the radical absorbance capacity (ORAC). For a more

direct measure of HDL-C antioxidant activity, levels of PON-1 could be measured, since

PON1 is the HDL enzyme component which some studies have reported to be

responsible for the antioxidative activity of HDL (13, 74, 75). Additionally, a larger sample

size in future studies will allow for greater power to detect differences in oxidative

markers, as well as the selection of a reference treatment in which MUFA would replace

carbohydrate sources without antioxidant activity in order to detect of the activity of

MUFAs.

HDL-C has also been recognized for its antithrombotic activities and studies done

with recombinant HDL-C or apoA1 have demonstrated regression of plaques and

improvements in atherosclerosis. Therefore, it would be interesting to conduct a larger

64

trial to try to raise HDL-C and apoA1 in subjects, for example with acute coronary

syndrome, and to include such measurements as intravascular ultrasound (IVUS) as a

measure of atheroma volume for which administration of recombinant apoA1Milano has

demonstrated significant improvement (88). This would allow for detection of quality of

HDL-C and functionality of apoA1 to see if raised apoA1 can reach the effectiveness

seen in the mutated version, apoA1Milano. Other groups with low levels of HDL-C, such as

in diabetes or the metabolic syndrome, both of which are also accompanied by

elevations in triglyceride, would be potentially interesting groups to observe effects

because of their increased risk of CVD (295).

65

7. Summary

66

7. Summary In carrying out this thesis the aim was to answer the objectives. We have

demonstrated the following:

1. HDL-C and apoA1 were significantly increased on the high-MUFA versus the

low-MUFA dietary portfolio without compromising the lipid-lowering effectiveness

of the diet.

2. Although there was no treatment difference between the high- and low-MUFA

dietary portfolio groups, in the subject group as a whole, there was a significant

increase in protein thiols and reductions in both CD and TBARS in the LDL

fraction, indicating reduced oxidative damage to serum proteins and lipids.

3. Both dietary portfolio treatments significantly reduced LDL-C and apoB

irrespective of MUFA intake.

4. Total:HDL-C ratio, as well as apoB:A1 ratio were significantly reduced on the

high-MUFA versus the low-MUFA dietary portfolio.

67

8. References 1. Research CIoH. Research About - Cardiovascular Health. Canadian Institutes of

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oxidative stress and risk of cardiovascular disease ......soy protein, which have also demonstrated...

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